Compositions and methods for treatment

ABSTRACT

The present invention provides compounds and methods for the treatment of LFA-1 mediated diseases. In particular, LFA-1 antagonists are described herein and these antagonists are used in the treatment of LFA-1 mediated diseases. One aspect of the invention provides for diagnosis of an LFA-1 mediated disease and administration of a LFA-1 antagonist, after the patient is diagnosed with a LFA-1 mediated disease. In some embodiments, the LFA-1 mediated diseases treated are dry eye disorders. Also provided herein are methods for identifying compounds which are LFA-1 antagonists.

CROSS-REFERENCE

This application is a Continuation application under 35 U.S.C. §120 ofU.S. application Ser. No. 14/610,182, filed Jan. 30, 2015, which is aContinuation application under 35 U.S.C. §120 of U.S. application Ser.No. 14/045,131, filed Oct. 3, 2013, now U.S. Pat. No. 9,045,457, whichis a Divisional application under 35 U.S.C. §120 of U.S. applicationSer. No. 13/011,760, filed Jan. 21, 2011, now U.S. Pat. No. 8,771,715,which is Continuation application under 35 U.S.C. §120 of U.S.application Ser. No. 12/508,367, filed Jul. 23, 2009, now U.S. Pat. No.8,084,047, and a Continuation application under 35 U.S.C. §120 of U.S.application Ser. No. 12/508,311, filed Jul. 23, 2009, each of which is aContinuation application under 35 U.S.C. §120 of U.S. application Ser.No. 11/436,906, filed May 17, 2006, now a U.S. Pat. No. 8,168,655, whichclaims the benefit under 35 U.S.C. §119(e) of U.S. Provisional PatentApplication No. 60/681,684, filed May 17, 2005; U.S. ProvisionalApplication No. 60/681,722, filed May 17, 2005; U.S. ProvisionalApplication No. 60/681,772, filed May 17, 2005, and U.S. ProvisionalApplication No. 60/681,723, filed May 17, 2005; each of which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

An ophthalmological disorder, dry eye, is a common complaint ofophthalmic patients. Unaddressed conditions of dry eye can lead toerosion and abrasion of the epithelial cell surface of the cornea,raising susceptibility to infection. Progression of the disease can leadto ulceration of the cornea, even loss of sight.

A variety of irritants, injuries, and medical conditions predisposeindividuals to initiation of decreased lacrimal gland secretionresulting in deficient levels of aqueous tears protecting and nourishingthe surface of the eye. There are environmental factors such as highaltitudes, arid and windy climates, air pollution, desiccated air fromcentral heat and central air conditioning, and exposure to cigarettesmoke which can establish and/or enhance deterioration of the quantityand quality of tear production. Even extensive computer use can be acontributing factor as studies have shown significantly decreasedblinking rates for users concentrating their attention on computerscreens. Some advances in eye care, starting with the introduction ofcontact lenses, and currently, the popularity of the LASIK procedure forvision correction, have contributed to the recent growth of subjectnumbers with dry eye. Use of contact lenses results in absorption oftear film by the lens, with resultant physical irritation of theconjunctiva in the eyelids. LASIK can have a secondary effect of eyeinjury as nerves often can be severed or ablated during laser refractivesurgery, which can lead to at least temporary dry eye syndrome ofseveral months duration.

Disease and some physical conditions can predispose individuals to dryeye disorder, including; allergies, diabetes, lacrimal gland deficiency,lupus, Parkinson's disease, Sjogren's syndrome, rheumatoid arthritis,rosacea, and others. Medications for other diseases may cause orexacerbate dry eye disorders, including diuretics, antidepressants,allergy medications, birth control pills, decongestants and others.

Age related changes may induce or exacerbate dry eye as well. Postmenopausal women experience changes in hormonal levels that caninstigate or worsen dry eye, and thyroid imbalances may cause similarchanges. Finally, aging itself can cause a reduction in lipid productionwith resultant dry eye.

Until recently, therapeutic interventions were limited to palliativemeasures to increase the moisture level of the eye. This is mostfrequently achieved with instillation of fluids which act as artificialtears. These fluids are often solutions which are instilled once orseveral times a day. For more severe cases of dry eye, artificial tearsolutions which incorporate a thickener or ocular gels can enhance theamount of film retained on the eye. Alternatively, several night-timeointment therapies are available. The thickened solutions, gels, andointments suffer from the limitation that vision can be impairedsignificantly upon application, rendering them less useful to theaverage subject who may require numerous applications during theirwaking, active hours. Another palliative intervention is theinstallation of temporary punctal occlusions, or even surgical closureof the normal drainage route of tears into the nasal cavity adjacent tothe eye.

However, none of these interventions are effective in the treatment ofthis disorder. Hence, it is desirable to develop agents whicheffectively treat dry eye, preferably with minimal side effects.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides methods for treatment ofinflammatory disorders mediated by LFA-1 by administering an effectiveamount of an antagonist of LFA-1 by itself or in combination with othertherapeutic agents to a subject. In some embodiments of the invention,diseases in which the anti-LFA-1 antibody, Raptiva, has showntherapeutic effect or effect on inflammatory cells in the diseasedtissue are diseases that are treated by the LFA-1 compounds of thepresent invention. Patients with immune mediated allergic diseasesincluding rhinitis may be treated with the compounds of the invention toreduce the inflammation associated with LFA-1 mediated immune and/orallergic responses. In some embodiments, a local administration of thecompounds of the invention, delivered via the mouth or nose as a mistedsolution or dispersed powder is useful in the treatment of asthma orother LFA-1 mediated pulmonary inflammatory diseases. In someembodiments, a cream formulation of the compounds of the invention isuseful in the local delivery of a LFA-1 antagonist to the skin indermatologic diseases mediated by LFA-1 such as eczema and psoriasis. Insome embodiments, an oral formulation of a LFA-1 antagonist which isknown to be poorly absorbed at the systemic level is administered by theoral route in animal studies is useful for local topical deliver ofLFA-1 antagonists in the treatment of inflammatory diseases of thegastrointestinal (GI) tract, including Crohn's disease and irritablebowel syndrome, or other GI disease mediated by LFA-1 or other leucocyteintegrins including VLA4 and Mac-1.

In some embodiments, the disorder that is mediated by LFA-1 is an eyedisorder. In some embodiments the inflammatory disorder that is mediatedby LFA-1 is dry eye. In particular, the methods of the present inventionare useful for treatment of dry eye syndrome. This syndrome encompassessymptoms caused by: keratoconjunctivitis sicca, Sjorgen's syndrome,corneal injury, age-related dry eye, Stevens-Johnson syndrome,congenital alachrima, pharmacological side effects, infection, Riley-Daysyndrome, conjunctival fibrosis, eye stress, glandular and tissuedestruction, ocular cicatrical pemphogoid, blepharitis, autoimmune andother immunodeficient disorders, allergies, diabetes, lacrimal glanddeficiency, lupus, Parkinson's disease, Sjogren's syndrome, rheumatoidarthritis, rosacea, environmental exposure to excessively dry air,airborne particulates, smoke, and smog and inability to blink, amongstothers. Many patients suffering from dry eye may also have an underlyingautoimmune disease, Sjogren's syndrome. Currently recognized diagnosticcriteria for patient identification include clinical signs and symptomsof dry mouth. The compounds of the present invention may be useful intreating this symptom, in formulations of mouthwash or lozenges. A skincream applied to the outer surface of the eyelids thus delivering aLFA-1 antagonist across the eyelid to the inner lining of the eyelid andthe intervening conjunctival tissue and accessory lacrimal glands isdesirable in treating LFA-1 mediated inflammation of the eyelid and eye,particularly in the treatment of dry eye.

Another aspect of the present invention provides pharmaceuticalcompositions which comprise a LFA-1 antagonist for administration in themethods of treatment of inflammatory disorders mediated by LFA-1. Insome embodiments the inflammatory disorder mediated by LFA-1 is an eyedisorder for which pharmaceutical compositions which comprise a LFA-1antagonist are provided. In some embodiments the inflammatory disordermediated by LFA-1 is dry eye, for which pharmaceutical compositionswhich comprise a LFA-1 antagonist have been provided. It is furtherprovided that the compositions may further comprise another therapeuticagent to be co-administered either in the same formulation orseparately. In some embodiments, the pharmaceutical compositions areadministered orally, via injection, intranasally, via inhalation,rectally, topically, via instillation to the ocular surface, ortransdermally.

In another aspect, the present invention provides formulations for thecompositions which are adminstered in the methods of treatment ofinflammatory disorders mediated by LFA-1. In some embodiments,gastro-retentive formulations of compositions are provided foradministration to treat inflammatory disorders mediated by LFA-1. Insome embodiments, gastro-retentive formulations of compositions areprovided for administration to treat eye disorders which areinflammatory disorders mediated by LFA-1. In some embodiments, ocularformulations of compositions are provided for administration to treatdry eye which is the inflammatory disorder mediated by LFA-1. In someembodiments, ocular formulations of compositions are provided foradministration to treat inflammatory disorders mediated by LFA-1. Insome embodiments, formulations of compositions are provided foradministration to treat inflammatory disorders mediated by LFA-1, whichare solutions, creams, powders, suspensions, mists, gels, solids, andthe like. Controlled release formulations are also provided for in someembodiments of the invention. In some embodiments of the invention, thecompounds of the invention are formulated as prodrugs.

In another aspect, compounds are provided for use in the methods of theinvention. Compounds that are useful in the methods of the inventioninclude antibodies, fragments of antibodies, polypeptides, peptides,polymers, and organic small molecules. In another an embodiment of themethod of the present invention, Raptiva is used in an ocularformulation to treat dry eye.

One aspect of the invention combines a diagnostic with a method oftreatment with an LFA-1 antagonist. In one embodiment, a diagnostic testfor Sjorgren's is performed and after a diagnosis of the disease ismade, the patient is administered an LFA-1 antagonist as describedherein. In another embodiment, a diagnostic test for dry eye isperformed and after a diagnosis of dry eye is made, the patient isadministered an LFA-1 antagonist as described herein.

The compounds provided herein are administered to increase tear or mucinproduction to a subject suffering from an inflammatory disorder mediatedby LFA-1. Preferably, the inflammatory disorder treated is an eyedisorder. Even more preferably, the inflammatory disorder is dry eye.

In another aspect, a method for identifying inhibitors of the LFA-1:ICAM-1 interaction is provided. In some embodiments, the inhibitors areidentified as being directly competitive with ICAM-1 binding to LFA-1 atthe αL subunit of LFA-1. In some embodiments, the method utilizescompetitive binding experiments to identify antagonists of the LFA-1:ICAM-1 interaction. In some embodiments, labeled probe molecules whichare known to bind at metal ion dependent adhesion site of theLFA-1:ICAM-1 interaction on the αL subunit of LFA-1 are employed.

In another aspect a method of identifying useful pharmaceutical agentsfor human disease is described using the pattern of the inhibition ofcell growth by siRNA (small interfering RNA sequences) directed againsta cellular target involved in cell growth and human disease to identifycompounds with a similar pattern of cell growth inhibition in a group ofcultured cell lines. The methods of this invention can also be used toidentify useful inhibitors of LFA-1, the B-cell receptor BR3, Grb2 (aprotein downstream of growth factor receptors in signaling cascades) andother protein targets inside and outside of cells. In another embodimentof this invention, the identification of compounds which fit an activitypattern opposite of the inhibition of cell growth by siRNA can bestimulants of cell growth useful in diseases and conditions of slow cellgrowth. Enhanced cell growth could be useful in wound healing and otherclinical settings. In another embodiment of this invention, this methoduses siRNA cellular activity data for target or selection of targets bysearching public and/or proprietary databases of compound cellularactivity for a pattern of similar cellular activity in response to acompound or collection of compounds as a method to identify compoundsuseful in the identification of a human pharmaceutical

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 depicts rolling, adhesion of leukocytes and transendothelialmigration resulting from LFA-1:ICAM-1 interaction.

FIG. 2 depicts antigen activation of the LFA-1:ICAM-1 interaction.

FIG. 3 depicts co-stimulatory function of the LFA-1:ICAM-1 interaction.

FIG. 4 depicts small molecule antagonists useful in the methods ofidentification.

FIG. 5 depicts Table 1 showing cation dependence of small moleculeantagonists for LFA-1.

FIG. 6 depicts SDS-PAGE analysis of compound 5 crosslinked LFA-1.

FIG. 7 depicts binding of compound 2B and ICAM-1-Ig to 293 cellsexpressing wild type LFA-1 or LFA-1 lacking the I domain.

FIG. 8 depicts antagonist competition by compounds 2A, 3, A-286982 andsICAM-1 in the LFA-1/ICAM-1 and LFA-1/small molecule ELISAs.

FIG. 9 depicts correlation of IC50 values from antagonist competition inthe LFA-1/ICAM-1 and LFA-1/small molecule ELISAs.

FIG. 10 depicts effect of antagonists on ligand binding in theLFA-1/ICAM-1 and LFA-1/small molecule ELISAs.

FIG. 11 depicts Schild regressions of sICAM-1 and compound 3 antagonism.

FIG. 12 depicts flow diagram of the discovery of potent inhibitors ofcell growth for the treatment of human cancer and inflammation.

DETAILED DESCRIPTION OF THE INVENTION I. Interaction of Leukointegrinsand Adhesion Receptors: Biology and Diseases

A first aspect of the present invention is methods for the treatment ofthe inflammatory component of immune and other disorders. In particular,the methods described herein are useful for the treatment of leukocytemediated inflammation. This component plays a role in initiating andadvancing inflammation in selected diseases, such as psoriasis, eczema,asthma, dermatitis, rheumatoid arthritis, systemic lupus erythematosis(SLE), multiple sclerosis, responses associated with inflammatory boweldisease, Reynaud's syndrome, Sjorgen's disease, juvenile onset diabetes,diabetes mellitus, granulomatosis, CNS inflammatory disorder, multipleorgan injury disease, all types of transplantations, including graftversus host or host versus graft disease, HIV and rhinovirus infections,and atherosclerosis amongst other diseases.

A preferred embodiment of this invention is for the treatment of eyedisorders. In particular, the methods of the present invention areuseful for treatment of dry eye syndrome. This syndrome encompassessymptoms caused by: keratoconjunctivitis sicca, Sjorgen's syndrome,corneal injury, age-related dry eye, Stevens-Johnson syndrome,congenital alachrima, pharmacological side effects, infection, Riley-Daysyndrome, conjunctival fibrosis, eye stress, glandular and tissuedestruction, ocular cicatrical pemphogoid, blepharitis, autoimmune andother immunodeficient disorders, allergies, diabetes, lacrimal glanddeficiency, lupus, Parkinson's disease, Sjogren's syndrome, rheumatoidarthritis, rosacea, environmental exposure to excessively dry air,airborne particulates, smoke, and smog and inability to blink, amongstothers.

Not intending to limit the mechanism of action, the methods of thepresent invention involve the inhibition of initiation and progressionof inflammation related disease by inhibiting the interaction betweenLFA-1 and ICAM-1. LFA-1 and ICAM-1 are molecules with extracellularreceptor domains which are involved in the process oflymphocyte/leukocyte migration and proliferation, leading to a cascadeof inflammatory responses. In preferred embodiments, such methodsprovide anti-inflammatory effects in-vitro and in-vivo, e.g., asdescribed in more detail below, and are useful in the treatment ofinflammation mediated diseases, and in particular, dry eye disease.

Human blood contains white blood cells (leukocytes) which are furtherclassified as neutrophils, lymphocytes (with B- and T-subtypes),monocytes, eosinophils, and basophils. Several of these classes ofleukocytes, neutrophils, eosinophils, basophils and lymphocytes, areinvolved in inflammatory disorders. LFA-1 is one of a group ofleucointegrins which are expressed on most leucocytes, and is consideredto be the lymphoid integrin which interacts with a number of ICAMs asligands Disrupting these interactions, and thus the immune/inflammatoryresponse provides for reduction of inflammation, in particular,inflammation of the eye.

For example, ICAM-1 (CD54) is a member of the ICAM family of adhesionreceptors (ICAM-1, ICAM-2, ICAM-3, ICAM-4) in the immunoglobulin proteinsuper family, and is expressed on activated leucocytes, dermalfibroblasts, and endothelial cells. See Krensky, A. M.; Sanchez-Madrid,F.; Robbins, E.; Nagy, J. A.; Springer, T. A. Burakoff, S. J. “Thefunctional significance, distribution, and structure of LFA-1, LFA-2,and LFA-3: cell surface antigens associated with CTL-targetinteractions.” 1983 J. Immunol. 131, 611-616. It is normally expressedon the endothelial cells lining the vasculature, and is upregulated uponexposure to cytokines such as IL-1, LPS and TNF duringimmune/inflammatory initiation.

Research conducted over the last decade has helped elucidate themolecular events involved in the movement and activation of cells in theimmune system, focusing on cell-to-cell triggering interactions withinthe cascade. See Springer, T. A. “Adhesion receptors of the immunesystem.” Nature, 1990, 346, 425-434. The interaction of IntercellularAdhesion Molecules (ICAMs) with leukointegrins plays a role in thefunctioning of the immune system. It is believed that immune processessuch as antigen presentation, T-cell mediated cytotoxicity and leukocytetransendothelial migration (diapedesis) require cellular adhesionmediated by ICAMs interacting with leukointegrins. See Kishimoto, T. K.;Rothlein; R. R. “Integrins, ICAMs, and selectins: role and regulation ofadhesion molecules in neutrophil recruitment to inflammatory sites.”Adv. Pharmacol. 1994, 25, 117-138 and Diamond, M.; Springer, T. A. “Thedynamic regulation of integrin adhesiveness.” Current Biology, 1994, 4,506-532.

The interaction of ICAM-1 and LFA-1 (also referred to as α_(L)β₂ andCD11a/CD18) has been shown to be involved in the processes of adhesion,leukocyte transendothelial migration, migration to sites of injury, andproliferation of lymphocytes at the activated target site, as shown inFIG. 1. For example, it is presently believed that prior to leukocytetransendothelial migration, a component of the inflammatory response,the presence of cytokines/chemokines activate integrins constitutivelyexpressed on leukocytes. Blood vessel endothelial cells also upregulateICAM-1 in response to the presence of the same cytokines/chemokines. Asrolling leukocytes approach activated endothelial cells, their progressis first slowed by these upregulated ICAM-1 receptors. This is followedby a ligand/receptor interaction between LFA-1 and ICAM-1, expressed onblood vessel endothelial cell surfaces, which arrests the lymphocytefrom rolling further. The lymphocyte then flattens, and transvasationtakes place. This process is of importance both in lymphocytetransmigration through vascular endothelial as well as lymphocytetrafficking from peripheral blood to lymph nodes.

LFA-1 plays a role in creating and maintaining the immunologicalsynapse, which may be defined as the physical structure of theinteracting surfaces of T cells and Antigen Presenting Cells (APCs), asshown in FIG. 2. LFA-1 stabilizes T-cell engagement with the APC, andthus leads to activation of T cells. The interaction of LFA-1 and ICAM-1also appears to provide co-stimulatory signals to resting T cells, asshown in FIG. 3. CD4+ T-cell proliferation and cytokine synthesis aremediated by this interaction as part of the inflammatory response.

Given the role that the interaction of ICAM-1 and LFA-1 plays inimmune/inflammatory response, it is desirable to modulate theseinteractions to achieve a desired therapeutic result (e g, inhibition ofthe interaction in the event of an overactive inflammatory response).Also, since LFA-1 has several ligand partners within the ICAM family(ICAM-1, ICAM-2 and ICAM-3), involving a number of signaling pathways,in some embodiments of the invention, it is desirable to modulate theseinteractions selectively. It has been demonstrated that the antagonismof the interaction between ICAMs and leukointegrins can be realized byagents directed against either component.

The methods and compositions described herein can modulate one or morecomponents of the pathways described herein. In addition to inhibitinginteraction between LFA-1 and ICAM-1, the methods and compositions ofthe present invention may also intervene in either earlier or laterportions of the inflammatory process as well. For example, upregulationof ICAM-1 or LFA-1 (activation) on endothelial cells or leukocytes,prior to tethering and transendothelial migration, may be modulated bythe methods and compositions described herein. The present invention maybe useful in modulating the expression of cytokines or chemokines thatactivate ICAM-1 and LFA-1 in the course of leukocyte trafficking, inmodulating the transport of the cytokines or chemokines, in preventingtransvasation of the arrested leukocyte, in modulating signalling viaother mechanisms that are involved in leukocyte proliferation at thesite of injury or inflammation, and the like.

II. Methods of Treatment

The term “subject” as used herein includes animals, in particular humansas well as other mammals. The methods generally involve theadministration of one or more drugs for the treatment of one or morediseases. Combinations of agents can be used to treat one disease ormultiple diseases or to modulate the side-effects of one or more agentsin the combination. The compounds described herein can be used incombination with other dry eye treatment agents. Also, the compounds ofthe invention can be used with drugs that cause dry eye as a sideeffect.

The term “treating” and its grammatical equivalents as used hereinincludes achieving a therapeutic benefit and/or a prophylactic benefit.By therapeutic benefit is meant eradication or amelioration of theunderlying disorder being treated. Also, a therapeutic benefit isachieved with the eradication or amelioration of one or more of thephysiological symptoms associated with the underlying disorder such thatan improvement is observed in the subject, notwithstanding that thesubject may still be afflicted with the underlying disorder. Forprophylactic benefit, the compositions may be administered to a subjectat risk of developing a particular disease, or to a subject reportingone or more of the physiological symptoms of a disease, even though adiagnosis of this disease may not have been made. The compositions maybe administered to a subject to prevent progression of physiologicalsymptoms or of the underlying disorder.

In some embodiments, the therapeutic agent is present in an amountsufficient to exert a therapeutic effect by an average of at least about5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, more than 90%, orsubstantially eliminate the disease or at least one of its underlyingsymptoms. Preferably the therapeutic effect is an effect oninflammation.

In some embodiments, the therapeutic agent is present in an amountsufficient to exert a therapeutic effect to reduce symptoms of dry eyeby an average of at least about 5, 10, 15, 20, 25, 30, 40, 50, 60, 70,80, 90, more than 90%, or substantially eliminate symptoms of dry eye.

In some embodiments, an effective amount of the therapeutic agent is adaily dose of about 1×10⁻¹¹, 1×10⁻¹⁰, 1×10⁻⁹, 1×10⁻⁸, 1×10⁻⁷, 1×10⁻⁶,1×10⁻⁵, 1×10⁻⁴, 1×10⁻³, 1×10⁻², 1×10⁻¹, 1, 1×10¹, 1×10² grams.

Administration of the therapeutic agent may be by any suitable means. Insome embodiments, the therapeutic agent is administered by oraladministration. In some embodiments, the therapeutic agent isadministered by transdermal administration. In some embodiments, thetherapeutic agent is administered by injection. In some embodiments, thetherapeutic agent is administered topically. If combinations of agentsare administered as separate compositions, they may be administered bythe same route or by different routes. If combinations of agents areadministered in a single composition, they may be administered by anysuitable route. In some embodiments, combinations of agents areadministered as a single composition by oral administration. In someembodiments, combinations of agents are administered as a singlecomposition by transdermal administration. In some embodiments, thecombinations of agent are administered as a single composition byinjection. In some embodiments, the combinations of agent areadministered as a single composition topically.

The method of the invention described herein is a method ofadministering an antagonist of LFA-1 to a subject to treat dry eye. Inparticular, the LFA-1 antagonist can modulate inflammation mediated byleukocytes. A preferred embodiment of the invention treats a subject byadministering an antagonist of LFA-1 to modulate inflammation associatedwith ocular inflammation. Another preferred embodiment of the method isto treat a subject with inflammation associated with dry eye syndrome byadministering an antagonist of LFA-1. An embodiment of the inventiontreats a subject with symptoms of dry eye due to allergies. Anembodiment of the invention treats a subject with symptoms of dry eyedisorder due to diabetes. An embodiment of the invention treats asubject with symptoms of dry eye disorder due to lacrimal glanddeficiency. An embodiment of the invention treats a subject withsymptoms of dry eye disorder due to lupus. An embodiment of theinvention treats a subject with symptoms of dry eye disorder due toParkinson's disease. An embodiment of the invention treats a subjectwith symptoms of dry eye disorder due to Sjogren's disease. Anembodiment of the invention treats a subject with symptoms of dry eyedisorder due to rheumatoid arthritis. An embodiment of the inventiontreats a subject with symptoms of dry eye disorder due to rosacea. Anembodiment of the invention treats a subject with symptoms of dry eyedisorder due to complications arising from LASIK therapy for visioncorrection. An embodiment of the invention treats a subject withsymptoms of dry eye disorder due to use of contact lenses. An embodimentof the invention treats a subject with symptoms of dry eye disorder dueto exposure to arid climates. An embodiment of the invention treats asubject with symptoms of dry eye disorder due to exposure to airpollution. An embodiment of the invention treats a subject with symptomsof dry eye disorder due to windy climates. An embodiment of theinvention treats a subject with symptoms of dry eye disorder due toexposure due to cigarette smoke. An embodiment of the invention treats asubject with symptoms of dry eye disorder due to keratoconjunctivitissicca. An embodiment of the invention treats a subject with symptoms ofdry eye disorder due to corneal injury. An embodiment of the inventiontreats a subject with symptoms of dry eye disorder due to conjunctivalfibrosis. An embodiment of the invention treats a subject with symptomsof dry eye disorder due to age-related dry eye. An embodiment of theinvention treats a subject with symptoms of dry eye disorder due toStevens-Johnson syndrome. An embodiment of the invention treats asubject with symptoms of dry eye disorder due to congenital alachrima.An embodiment of the invention treats a subject with symptoms of dry eyedisorder due to pharmacological side effects of other drugs being takenby the patient. An embodiment of the invention treats a subject withsymptoms of dry eye disorder due to infection. An embodiment of theinvention treats a subject with symptoms of dry eye disorder due toRiley-Day syndrome. An embodiment of the invention treats a subject withsymptoms of dry eye disorder due to eye stress, including that due tocomputer use. An embodiment of the invention treats a subject withsymptoms of dry eye disorder due to glandular and tissue destruction. Anembodiment of the invention treats a subject with symptoms of dry eyedisorder due to ocular cicatrical pemphogoid. An embodiment of theinvention treats a subject with symptoms of dry eye disorder due toblepharitis. An embodiment of the invention treats a subject withsymptoms of dry eye disorder due to autoimmune and other immunodeficientdisorders. An embodiment of the invention treats a subject with symptomsof dry eye disorder due to an inability to blink. An embodiment of theinvention treats a subject with symptoms of psoriasis with a LFA-1antagonist of the method. An embodiment of the invention treats asubject with symptoms of eczema with a LFA-1 antagonist of the method.An embodiment of the invention treats a subject with symptoms of lupuswith a LFA-1 antagonist of the method. An embodiment of the inventiontreats a subject with symptoms of Reynaud's syndrome with a LFA-1antagonist of the method. An embodiment of the invention treats asubject with symptoms of granulomatosis with a LFA-1 antagonist of themethod. An embodiment of the invention treats a subject with symptoms ofCNS inflammatory disorder with a LFA-1 antagonist of the method. Anembodiment of the invention treats a subject with symptoms of multipleorgan disease with a LFA-1 antagonist of the method. An embodiment ofthe invention treats a subject with symptoms of allergic rhinitis with aLFA-1 antagonist of the method. An embodiment of the invention treats asubject with symptoms of granulomatosis with a LFA-1 antagonist of themethod. An embodiment of the invention treats a subject with symptoms ofatherosclerosis with a LFA-1 antagonist of the method. An embodiment ofthe invention treats a subject with symptoms of graft versus hostdisease with a LFA-1 antagonist of the method. An embodiment of theinvention treats a subject with symptoms of host versus graft diseasewith a LFA-1 antagonist of the method. An embodiment of the inventiontreats a subject with symptoms of inflammatory response associated withtransplantation with a LFA-1 antagonist of the method. An embodiment ofthe invention treats a subject with symptoms of inflammatory boweldisease with a LFA-1 antagonist of the method. An embodiment of theinvention treats a subject with symptoms of juvenile onset diabetes witha LFA-1 antagonist of the method. An embodiment of the invention treatsa subject with symptoms of diabetes mellitus with a LFA-1 antagonist ofthe method. An embodiment of the invention treats a subject withsymptoms of multiple sclerosis with a LFA-1 antagonist of the method. Anembodiment of the invention treats a subject with symptoms of asthmawith a LFA-1 antagonist of the method. An embodiment of the inventiontreats a subject with symptoms of dermatitis with a LFA-1 antagonist ofthe method. An embodiment of the invention treats a subject withsymptoms of systemic lupus erythematosis with a LFA-1 antagonist of themethod. An embodiment of the invention treats a subject with symptoms ofHIV and rhinovirus infections with a LFA-1 antagonist of the method.

In some embodiments of the invention, diagnostic procedures will beemployed to identify a subject in need of treatment by the method of theinvention. Fluorescein staining of the cornea is used to diagnosesymptoms of dry eye disorder. Rose Bengal staining of the cornea is usedto diagnose symptoms of dry eye disorder. Corneal sensitivity is used todiagnose symptoms of dry eye disorder. Tear breakup time (BUT) is usedto diagnose symptoms of dry eye disorder. Schirmer test with anesthesiais used to diagnose symptoms of dry eye disorder. Schirmer test analysisis used to diagnose symptoms of dry eye disorder. Impression cytology isused to diagnose symptoms of dry eye disorder. Subjective dry eyesymptoms are used to diagnose symptoms of dry eye disorder. Tear flowanalysis is used to diagnose symptoms of dry eye disorder.Immunohistochemical methods, including but not limited to humanleukocyte antigen II (HLA-DR), are used to diagnose symptoms of dry eyedisorder. Antinuclear antibody test (ANA) or fluorescent antinuclearantibody test (FANA) is used to diagnose symptoms of dry eye disorder.Ocular evaporation is used to diagnose symptoms of dry eye disorder.Infrared meibography is used to diagnose symptoms of dry eye disorder.Tandem scanning confocal microscopy (TSCM) is used to diagnose symptomsof dry eye disorder. This is an exemplary list of procedures that may beused to diagnose symptoms of dry eye and is in no way limiting.

The antagonist of the method of the invention may be an antibody,fragment of an antibody, peptide or small molecule. In preferredembodiments, the LFA-1 antagonist used is a peptide which is not anantibody. The antagonist of the method is a therapeutic agent.

Many therapeutic indications for LFA-1 antagonists require chronictherapy; therefore, small molecule inhibitors of the LFA-1/ICAM-1interaction are one group of preferred embodiments of this invention asthey have the potential for oral administration as well as a loweredcost of goods.

A further preferred embodiment is a method of treating dry eye diseaseusing therapeutic agents which are suitable for formulation andadministration as ocular therapeutics.

Another aspect of the present invention is described herein and below, amethod of comparison of the binding of ICAM-1 and antagonists which canbe utilized to identify antibodies, antibody fragments, peptides, andsmall molecules as antagonists of the LFA-1: ICAM-1 interaction. SeeGadek et al. 2002. The method is described in terms of identifying smallmolecule antagonists. However, it should not be interpreted as limitingthe method in any manner to exclude its use in identifying largermolecule types of inhibitors of LFA-1, such as antibodies, fragments ofantibodies or peptides.

This method comprises choosing one or more of the following steps aspart of the process of identifying an antagonist as a directlycompetitive inhibitor of LFA-1: (a) competition experiments utilizingfull length wild type LFA-1 comparing the binding of potentialantagonistic agents to that of sICAM-1 (the extracellular domains ofLFA-1's native ligand and a competitive LFA-1/ICAM-1 inhibitor) andA-286982 (an allosteric LFA-1/ICAM-1 inhibitor known to bind to the I(inserted) domain allosteric site (IDAS)). See Liu, G.; Huth, J. R.;Olejniczak, E. T.; Mendoza, R.; DeVries, P.; Leitza, S.; Reilly; E. B.,Okasinski; G. F.; Fesik, S. W.; and von Geldern, T. W. 2001. “Novelp-arylthio cinnamides as antagonists of leukocyte function-associatedantigen-1/intracellular adhesion molecule-1 interaction. 2. Mechanism ofinhibition and structure-based improvement of pharmaceuticalproperties.” J. Med. Chem., 44, 1202-1210)), (b) binding studies ofpotential antagonistic agents and ICAM-1 with a LFA-1 mutant, and (c)chemical crosslinking studies. The ICAM-1 binding site targeted hereinhas previously been localized to include the metal ion dependentadhesion site (MIDAS) motif within the I domain of the LFA-1 α subunit.See Shimaoka, M., Xiao, T., Liu, J.-H., Yang, Y., Dong, Y., Jun, C-D.,McCormack, A. Zhang, R., Joachimiak, A., Takagi, J., Wang, J.-H., andSpringer, T. A. 2003 “Structures of the alpha L I domain and its complexwith ICAM-1 reveal a shape-shifting pathway for integrin regulation”Cell 2003, 99-111. Antagonists that inhibit ICAM-1 binding to LFA-1 bydirect competition for a common high affinity binding site on LFA-1 canbe identified using one or more steps of this method.

A. Antibodies as Therapeutic Agents

Several suitable antibodies are known in the art. Blocking of the CAMs,such as for example ICAM-1, or the leukointegrins, such as for example,LFA-1, by antibodies directed against either or both of these moleculescan inhibit inflammatory response. Previous studies have investigatedthe effects of anti-CD11a MAbs on many T-cell-dependent immune functionsin vitro and a number of immune responses in vivo. In vitro, anti-CD11aMAbs inhibit T-cell activation (See Kuypers T. W., Roos D. 1989“Leukocyte membrane adhesion proteins LFA-1, CR3 and p150,95: a reviewof functional and regulatory aspects” Res. Immunol., 140:461-465;Fischer A, Durandy A, Sterkers G, Griscelli C. 1986 “Role of the LFA-1molecule in cellular interactions required for antibody production inhumans” J. Immunol., 136, 3198; target cell lysis by cytotoxicT-lymphocytes (Krensky et al., supra), formation of immune conjugates(Sanders V M, Snyder J M, Uhr J W, Vitetta E S., “Characterization ofthe physical interaction between antigen-specific B and T cells”. J.Immunol., 137:2395 (1986); Mentzer S J, Gromkowski S H, Krensky A M,Burakoff S J, Martz E. 1985 “LFA-1 membrane molecule in the regulationof homotypic adhesions of human B lymphocytesn” J. Immunol., 135:9), andthe adhesion of T-cells to vascular endothelium (Lo S K, Van Seventer GA, Levin S M, Wright S D., Two leukocyte receptors (CD11a/CD18 andCD11b/CD18) mediate transient adhesion to endothelium by binding todifferent ligands., J. Immunol., 143:3325 (1989)). Two anti-CD11a MAbs,HI 111, and G43-25B are available from Pharmingen/BD Biosciences.Additionally, a study including F8.8, CBR LFA 1/9, BLS, May.035, TS1/11,TS1/12, TS 1/22, TS2/14, 25-3-1, MHM2 and efalizumab evaluated the rangeof binding sites on LFA-1 these antibodies occupied. See Lu, C;Shimaoka, M.; Salas, A.; Springer, T. A. 2004, “The Binding Sites forCompetitive Antagonistic, Allosteric Antagonistic, and AgonisticAntibodies to the I Domain of Integrin LFA-1” J. Immun 173: 3972-3978and references therein.

The observation that LFA-1:ICAM-1 interaction is necessary to optimizeT-cell function in vitro, and that anti-CD11a MAbs induce tolerance toprotein antigens (Benjamin R J, Qin S X, Wise M P, Cobbold S P, WaldmannH. 1988 “Mechanisms of monoclonal antibody-facilitated toleranceinduction: a possible role for the CD4 (L3T4) and CD11a (LFA-1)molecules in self-non-self discrimination” Eur. J. Immunol., 18:1079)and prolongs tumor graft survival in mice (Heagy W, Walterbangh C, MartzE. 1984 “Potent ability of anti-LFA-1 monoclonal antibody to prolongallograft survival” Transplantation, 37: 520-523) was the basis fortesting the MAbs to these molecules for prevention of graft rejection inhumans Experiments have also been carried out in primates. For example,based on experiments in monkeys, it has been suggested that a MAbdirected against ICAM-1 can prevent or even reverse kidney graftrejection (Cosimi et al., “Immunosuppression of Cynomolgus Recipients ofRenal Allografts by R6.5, a Monoclonal Antibody to IntercellularAdhesion Molecule-1,” in Springer et al. (eds.), Leukocyte AdhesionMolecules New York: Springer, (1988), p. 274; Cosimi et al., J.Immunology, 144:4604-4612 (1990)). Furthermore, the in vivoadministration of anti-CD11a MAb to cynomolgus monkeys prolonged skinallograft survival See Berlin et al., Transplantation, 53: 840-849(1992).

B. Small Molecules

Peptides have been investigated for use in reducing the interaction ofLFA-1 with ICAM-1. Polypeptides that do not contain an Fc region of anIgG are described in U.S. Pat. No. 5,747,035, which can be used to treatLFA-1 mediated disorders, in particular dry eye. Use of dual peptides,the first a modulator of ICAM-1 and the second a blocking peptide with asequence obtained from LFA-1 is described in U.S. Pat. No. 5,843,885 toreduce the interactions between LFA-1 and ICAM-1. Cyclic peptides havebeen described in U.S. Pat. No. 6,630,447 as inhibitors of the LFA-1:ICAM-1 interaction.

Small molecule antagonists include statins which bind to the CD11adomain of LFA-1. See Kallen, J., Welzenbach, K., Ramage, P. Geyl, D.Kriwacki, R., Legge, G., Cottens, S., Weitz-Schmidt, G., and Hommel, U.1999. “Structural basis for LFA-1 inhibition upon lovastatin binding tothe CD11a I-domain”, J. Mol. Biol., 292: 1-9; and Weitz-Schmidt, G.,Welzenbach, K., Brinkmann, V, Kamata, T, Kallen, J., Bruns, C., Cottens,S., Takada, Y, and Hommel, U. 2001. Statins selectively inhibitleukocyte function antigen-1 by binding to a novel regulatory integrinsite, Nature Med., 7: 687-692; and Frenette, P. S. 2001. “Locking aleukocyte integrin with statins”, N. Engl. J. Med., 345: 1419-1421.Molecules derived from the mevinolin/compactin motif also show activityagainst LFA-1. See Welzenbach, K., Hommel, U., and Weitz-Schmidt, G.2002. “Small molecule inhibitors induce conformational changes in the Idomain and the I-like domain of Lymphocyte Function-AssociatedAntigen-1”, J. Biol. Chem., 277: 10590-10598, and U.S. Pat. No.6,630,492.

A family of hydantoin-based inhibitors can also be used as antagonists.See Kelly, T. A., Jeanfavre, D. D., McNeil, D. W., Woska, J. R. Jr.,Reilly, P. L., Mainolfi, E. A., Kishimoto, K. M., Nabozny, G. H.,Zinter, R., Bormann, B.-J., and Rothlein, R. 1999. “Cutting edge: asmall molecule antagonist of LFA-1-mediated cell adhesion”, J. Immunol.,163: 5173-5177. These compounds are believed to be allosteric inhibitorsof LFA-1.

A family of novel p-arylthio cinnamides can act as antagonists of LFA-1.See Liu, G.; Link, J. T.; Pei, Z.; Reilly, E. B.; Nguyen, B.; Marsh, K.C.; Okasinski, G. F.; von Geldern, T. W.; Ormes, M.; Fowler, K.;Gallatin, M. 2000 “Discovery of novel p-arylthio cinnamides asantagonists of leukocyte function-associated antigen-1/intracellularadhesion molecule-1 interaction. 1. Identification of an additionalbinding pocket based on an anilino diaryl sulfide lead.” J. Med. Chem.43, 4015-4030.

Other families of small molecule inhibitors are disclosed inpublications (See Gadek, T. R., Burdick, D. J., McDowell, R. S.,Stanley, M. S., Marsters, J. C. Jr., Paris, K. J., Oare, D. A.,Reynolds, M. E., Ladner, C., Zioncheck, K. A., Lee, W. P., Gribling, P.,Dennis, M. S., Skelton, N. J., Tumas, D. B., Clark, K. R., Keating, S.M., Beresini, M. H., Tilley, J. W., Presta, L. G., and Bodary, S. C.2002. “Generation of an LFA-1 antagonist by the transfer of the ICAM-1immunoregulatory epitope to a small molecule” Science, 295: 1086-1089and online supplementary material.) and in patents, including U.S. Pat.No. 6,872,735, U.S. Pat. No. 6,667,318, U.S. Pat. No. 6,803,384, U.S.Pat. No. 6,515,124, U.S. Pat. No. 6,331,640, and patent applications,including: U.S. 20020119994. U.S. 20040058968, U.S. 20050080119,WO99/49856, WO00/21920, WO01/58853, WO02/59114, WO05/044817, and others.The contents of all the cited references are incorporated in theirentirety by reference.

In some embodiments, the compounds described herein are used incombination with restasis (Cyclosporine A). The compounds of theinvention can also be used to increase mucin production and/or tearproduction. Thus, the compounds of the present invention can offeradditional relief beyond decreasing inflammation and by also increasingthe mucin production that makes up a portion of tear film.

The interaction of LFA-1 and ICAMs are known to be involved in variousautoimmune and inflammatory diseases, particularly those withinvolvement of lymphocytic (T- or B-cell), dendritic, monocytic cellsexpressing LFA-1 on their surface as part of the inflammatory componentof disease. LFA-1 antagonists can be particularly useful in treatment ofthese diseases because the therapeutic target's expression in diseasedtissue is limited to infiltrating cells of the immune system. LFA-1 canblock the adhesion, migration, proliferation, and release ofinflammatory signals to surrounding tissue by immune system cells. Theanti-LFA-1 antibody, Raptiva, which has an effect on inflammatory cellsin diseased tissue may be used to treat dry eye.

Many patients suffering from dry eye may also have an underlyingautoimmune disease, Sjogren's syndrome. Currently recognized diagnosticcriteria include clinical signs and symptoms of Dry Mouth. The compoundsof the present invention may be useful in treating this symptom, informulations of mouthwash or lozenges. A lozenge incorporating thecompounds of the invention in a solid or waxy material may stimulatesalivary secretion while releasing the compound of the invention undersustained release.

Patients with immune mediated allergic diseases including rhinitis maybe treated with the compounds of the invention. For example, a LFA-1antagonist may be delivered locally to the nose, nasal passages, and/ornasal cavity to reduce the inflammation associated immune and/orallergic responses.

A local administration of the compounds of the invention, delivered viathe mouth or nose as a misted solution or dispersed powder may be usefulin the treatment of Asthma or other LFA-1 mediated pulmonaryinflammatory diseases.

A cream formulation of the compounds of the invention could be useful inthe local delivery of a LFA-1 antagonist to the skin in dermatologicdiseases mediated by LFA-1 such as eczema and psoriasis. Compoundsuseful in this regard include LFA-1 antagonists and their pro-drugswhich are transformed into the active drug in inflamed skin. A skincream applied to the outer surface of the eyelids thus delivering aLFA-1 antagonist across the eyelid to the inner lining of the eyelid andthe intervening conjunctival tissue and accessory lacrimal glands may bedesirable in treating LFA-1 mediated inflammation of the eyelid and eye,particularly in the treatment of dry eye.

An oral formulation of a LFA-1 antagonist which is known to be poorlyabsorbed at the systemic level by the oral route in animal studies maybe useful for local topical deliver of LFA-1 antagonists in thetreatment of inflammatory diseases of the gastrointestinal (GI) tract,including Crohn's disease and Irritable Bowel Syndrome, or other GIdisease mediated by LFA-1 or other leucocyte integrins including VLA4and Mac-1.

II. Compounds Useful in the Method A. Definitions

The term “aliphatic”, as used herein, includes both saturated andunsaturated, straight chain (unbranched) or branched aliphatichydrocarbons, which are optionally substituted with one or morefunctional groups. As will be appreciated by one of ordinary skill inthe art, “aliphatic” is intended herein to include, but is not limitedto, alkyl, alkenyl, alkynyl moieties. Thus, as used herein, the term“alkyl” includes straight and branched alkyl groups. An analogousconvention applies to other generic terms such as “alkenyl”, “alkynyl”and the like.

Furthermore, as used herein, the terms “alkyl”, “alkenyl”, “alkynyl”,and the like encompass both substituted and unsubstituted groups. Incertain embodiments, as used herein, “lower alkyl” is used to indicatethose alkyl groups (substituted, unsubstituted, branched or unbranched)having about 1-6 carbon atoms.

In certain embodiments, the alkyl, alkenyl and alkynyl groups employedin the invention contain about 1-20 aliphatic carbon atoms. In certainother embodiments, the alkyl, alkenyl, and alkynyl groups employed inthe invention contain about 1-10 aliphatic carbon atoms. In yet otherembodiments, the alkyl, alkenyl, and alkynyl groups employed in theinvention contain about 1-8 aliphatic carbon atoms. In still otherembodiments, the alkyl, alkenyl, and alkynyl groups employed in theinvention contain about 1-6 aliphatic carbon atoms. In yet otherembodiments, the alkyl, alkenyl, and alkynyl groups employed in theinvention contain about 1-4 carbon atoms. Illustrative aliphatic groupsthus include, but are not limited to, for example, methyl, ethyl,n-propyl, isopropyl, allyl, n-butyl, sec-butyl, isobutyl, tert-butyl,n-pentyl, sec-pentyl, isopentyl, tert-pentyl, n-hexyl, sec-hexyl,moieties and the like, which again, may bear one or more substituents.

Alkenyl groups include, but are not limited to, for example, ethenyl,propenyl, butenyl, and the like. Representative alkynyl groups include,but are not limited to, ethynyl, 2-propynyl and the like.

The term “lower alkylene” as used herein refers to a hydrocarbon chainwhich links together two other groups, i.e. is bonded to another groupat either end, for example methylene, ethylene, butylene and the like.Such a substituent is preferably from 1 to 10 carbons and morepreferably from 1 to 5 carbons. Such groups may be substituted,preferably with an amino, acetylamino (a lower alkylcarbonyl groupbonded via a nitrogen atom), or cyclo lower alkyl group. By the latteris meant a saturated hydrocarbon ring, preferably with a total of 3 to10 methylenes (inclusive of the attachment carbons), more preferably 3to 6.

The term “alicyclic”, as used herein, refers to compounds which combinethe properties of aliphatic and cyclic compounds and include but are notlimited to monocyclic, or polycyclic aliphatic hydrocarbons and bridgedcycloalkyl compounds, which are optionally substituted with one or morefunctional groups.

As will be appreciated by one of ordinary skill in the art, “alicyclic”is intended herein to include, but is not limited to, cycloalkyl,cycloalkenyl, and cycloalkynyl moieties, which are optionallysubstituted with one or more functional groups.

Illustrative alicyclic groups thus include, but are not limited to, forexample, cyclopropyl, —CH₂-cyclopropyl, cyclobutyl, —CH₂-cyclobutyl,cyclopentyl, —CH₂-cyclopentyl, cyclohexyl, —CH₂-cyclohexyl,cyclohexenylethyl, cyclohexanylethyl, norbornyl moieties and the like,which again, may bear one or more substituents.

The term “alkoxy” or “alkyloxy”, as used herein refers to a saturated orunsaturated parent molecular moiety through an oxygen atom. In certainembodiments, the alkyl group contains about 1-20 aliphatic carbon atoms.In certain other embodiments, the alkyl group contains about 1-10aliphatic carbon atoms. In yet other embodiments, the alkyl groupemployed in the invention contains about 1-8 aliphatic carbon atoms. Instill other embodiments, the alkyl group contains about 1-6 aliphaticcarbon atoms. In yet other embodiments, the alkyl group contains about1-4 aliphatic carbon atoms. Examples of alkoxy, include but are notlimited to, methoxy, ethoxy, isopropoxy, n-butoxy, i-butoxy, sec-butoxy,tert-butoxy, neopentoxy, n-hexloxy and the like.

The term “lower alkoxy” as used herein refers to a lower alkyl asdefined above which may be branched or unbranched as also defined aboveand which is bonded by an oxygen to another group (i.e. alkyl ethers).

The term “thioalkyl” as used herein refers to a saturated or unsaturated(i. e., S-alkenyl and S-alkynyl) group attached to the parent molecularmoiety through a sulfur atom. In certain embodiments, the alkyl groupcontains about 1-20 aliphatic carbon atoms. In certain otherembodiments, the alkyl group contains about 1-10 aliphatic carbon atoms.In yet other embodiments, the alkyl group employed in the inventioncontains about 1-8 aliphatic carbon atoms. In still other embodiments,the alkyl group contains about 1-6 aliphatic carbon atoms. In yet otherembodiments, the alkyl group contains about 1-4 aliphatic carbon atoms.Examples of thioalkyl include, but are not limited to, methylthio,ethylthio, propylthio, isopropylthio, n-butylthio, and the like.

The term “lower alkylthio” as used herein refers to a lower alkyl groupbonded through a divalent sulfur atom, for example, a methylmercapto oran isopropylmercapto group. By lower alkylenethio is meant such a groupwhich is bonded at each end.

The term “alkylamino” refers to a group having the structure-NHR′wherein R′ is alkyl, as defined herein. The term “aminoalkyl” refers toa group having the structure NH₂R′—, wherein as defined herein. Incertain embodiments, the alkyl group contains about 1-20 aliphaticcarbon atoms. In certain other embodiments, the alkyl group containsabout 1-10 aliphatic carbon atoms. In yet other embodiments, the alkylgroup employed in the invention contains about aliphatic carbon atoms.In still other embodiments, the alkyl group contains about 1-6 aliphaticcarbon atoms. In yet other embodiments, the alkyl group contains about1-4 aliphatic carbon atoms. Examples of alkylamino include, but are notlimited to, methylamino, and the like.

Some examples of substituents of the above-described aliphatic (andother) moieties of compounds of the invention include, but are notlimited to aliphatic; alicyclic; heteroaliphatic; heterocyclic;aromatic; heteroaromatic; aryl; heteroaryl; alkylaryl; heteroalkylaryl;alkylheteroaryl; heteroalkylheteroaryl; alkoxy; aryloxy; heteroalkoxy;heteroaryloxy; alkylthio; arylthio; heteroalkylthio; R_(x) independentlyincludes, but is not limited to, aliphatic, alicyclic, heteroaliphatic,heterocyclic, aryl, heteroaryl, alkylaryl, alkylheteroaryl,heteroalkylaryl or heteroalkylheteroaryl, wherein any of the aliphatic,alicyclic, heteroaliphatic, heterocyclic, alkylaryl, or alkylheteroarylsubstituents described above and herein may be substituted orunsubstituted, branched or unbranched, saturated or unsaturated, andwherein any of the aryl or heteroaryl substituents described above andherein may be substituted or unsubstituted. Additional examples ofgenerally applicable substituents are illustrated by the specificembodiments shown in the Examples that are described herein.

In general, the term “aromatic moiety”, as used herein, refers to astable mono- or polycyclic, unsaturated moiety having preferably 3-14carbon atoms, each of which may be substituted or unsubstituted. Incertain embodiments, the term “aromatic moiety” refers to a planar ringhaving p-orbitals perpendicular to the plane of the ring at each ringatom and satisfying the Huckel rule where the number of pi electrons inthe ring is (4n+2) wherein n is an integer. A mono- or polycyclic,unsaturated moiety that does not satisfy one or all of these criteriafor aromaticity is defined herein as “non-aromatic”, and is encompassedby the term “alicyclic”.

In general, the term “heteroaromatic moiety”, as used herein, refers toa stable mono- or polycyclic, unsaturated moiety having preferably 3-14carbon atoms, each of which may be substituted or unsubstituted; andcomprising at least one heteroatom selected from O, S, and N within thering in place of a ring carbon atom). In certain embodiments, the term“heteroaromatic moiety” refers to a planar ring comprising at least oneheteroatom, having p-orbitals perpendicular to the plane of the ring ateach ring atom, and satisfying the Huckel rule where the number of pielectrons in the ring is (4n+2) wherein n is an integer.

It will also be appreciated that aromatic and heteroaromatic moieties,as defined herein may be attached via an alkyl or heteroalkyl moiety andthus also include-(alkyl) aromatic, -(heteroalkyl) aromatic,-(heteroalkyl) heteroaromatic, and -(heteroalkyl) heteroaromaticmoieties. Thus, as used herein, the phrases“aromatic or heteroaromaticmoieties” and“aromatic, (heteroalkyl) aromatic, -(heteroalkyl)heteroaromatic, and (heteroalkyl) heteroaromatic” are interchangeable.Substituents include, but are not limited to, any of the previouslymentioned substituents, e., the substituents recited for aliphaticmoieties, or for other moieties as disclosed herein, resulting in theformation of a stable compound.

The term “aryl”, as used herein, does not differ significantly from thecommon meaning of the term in the art, and refers to an unsaturatedcyclic moiety comprising at least one aromatic ring. In certainembodiments, “aryl” refers to a mono- or bicyclic carbocyclic ringsystem having one or two aromatic rings including, but not limited to,phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl and the like.

The term “heteroaryl” as used herein, does not differ significantly fromthe common meaning of the term in the art, and refers to a cyclicaromatic radical having from five to ten ring atoms of which one ringatom is selected from S, and N; zero, one or two ring atoms areadditional heteroatoms independently selected from S, and N; and theremaining ring atoms are carbon, the radical being joined to the rest ofthe molecule via any of the ring atoms, such as, for example, pyridyl,pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl,oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl,quinolinyl, isoquinolinyl, and the like.

It will be appreciated that aryl and heteroaryl groups (includingbicyclic aryl groups) can be unsubstituted or substituted, whereinsubstitution includes replacement of one or more of the hydrogen atomsthereon independently with any one or more of the following moietiesincluding, but not limited to: aliphatic; alicyclic; heteroaliphatic;heterocyclic; aromatic; heteroaromatic; aryl; heteroaryl; alkylaryl;heteroalkylaryl; alkylheteroaryl; heteroalkylheteroaryl; alkoxy;aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio;heteroalkylthio; heteroarylthio; F; Cl; Br; I; —OH; —NO₂; —CN; —CF₃;—CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂; —CH₂SO₂CH₃; —C(═O)R_(x);—C(═O)N(R_(x))₂; —OC(═O)R_(x); —OCO₂R_(x); —OC(═O)N(R_(x))₂; —N(R_(x))₂;—S(O)₂R_(x); —NR_(x)(CO)R_(x) wherein each occurrence of R_(x)independently includes, but is not limited to, aliphatic, alicyclic,heteroaliphatic, heterocyclic, aromatic, heteroaromatic, aryl,heteroaryl, alkylaryl, alkylheteroaryl, heteroalkylaryl orheteroalkylheteroaryl wherein any of the aliphatic, alicyclic,heteroaliphatic, heterocyclic, alkylaryl, or alkylheteroarylsubstituents described above and herein may be substituted orunsubstituted, branched or unbranched, saturated or unsaturated, andwherein any of the aromatic, heteroaromatic, aryl, heteroaryl, -(alkyl)aryl or -(alkyl) heteroaryl substituents described above and herein maybe substituted or unsubstituted. Additionally, it will be appreciated,that any two adjacent groups taken together may represent a 4, 5, 6, or7-membered substituted or unsubstituted alicyclic or heterocyclicmoiety. Additional examples of generally applicable substituents areillustrated by the specific embodiments shown in the Examples that aredescribed herein.

The term “cycloalkyl”, as used herein, refers specifically to groupshaving three to seven, preferably three to ten carbon atoms. Suitablecycloalkyls include, but are not limited to cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl and the like, which, as in the caseof aliphatic, alicyclic, heteroaliphatic or heterocyclic moieties, mayoptionally be substituted with substituents including, but not limitedto aliphatic; alicyclic; heteroaliphatic; heterocyclic; aromatic;heteroaromatic; aryl; heteroaryl; alkylaryl; heteroalkylaryl;alkylheteroaryl; heteroalkylheteroaryl; alkoxy; aryloxy; heteroalkoxy;heteroaryloxy; alkylthio; heteroarylthio; F; Cl; Br; I; —OH; —NO₂; —CN;—CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂; —CH₂SO₂CH₃;—C(═O)R_(x); —C(═O)N(R_(x))₂; —OC(═O)R_(x); —OCO₂R_(x);—OC(═O)N(R_(x))₂; —N(R_(x))₂; —S(O)₂R_(x); —NR_(x)(CO)R_(x) wherein eachoccurrence of R_(x) independently includes, but is not limited to,aliphatic, alicyclic, heteroaliphatic, heterocyclic, aromatic,heteroaromatic, aryl, heteroaryl, alkylaryl, alkylheteroaryl,heteroalkylaryl or heteroalkylheteroaryl, wherein any of the aliphatic,alicyclic, heteroaliphatic, heterocyclic, alkylaryl, or alkylheteroarylsubstituents described above and herein may be substituted orunsubstituted, branched or unbranched, saturated or unsaturated, andwherein any of the aromatic, heteroaromatic, aryl or heteroarylsubstituents described above and herein may be substituted orunsubstituted. Additional examples of generally applicable substituentsare illustrated by the specific embodiments shown in the Examples thatare described herein.

The term “heteroaliphatic”, as used herein, refers to aliphatic moietiesin which one or more carbon atoms in the main chain have beensubstituted with a heteroatom. Thus, a heteroaliphatic group refers toan aliphatic chain which contains one or more oxygen, sulfur, nitrogen,phosphorus or silicon atoms, e. place of carbon atoms. Heteroaliphaticmoieties may be linear or branched, and saturated or unsaturated. Incertain embodiments, heteroaliphatic moieties are substituted byindependent replacement of one or more of the hydrogen atoms thereonwith one or more moieties including, but not limited to aliphatic;alicyclic; heteroaliphatic; heterocyclic; aromatic; heteroaromatic;aryl; heteroaryl; alkylaryl; alkylheteroaryl; alkoxy; aryloxy;heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroarylthio; F; Cl;Br; I; —OH; —NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH;—CH₂NH₂; —CH₂SO₂CH₃; —C(═O)R_(x); —C(═O)N(R_(x))₂; —OC(═O)R_(x);—OCO₂R_(x); —OC(═O)N(R_(x))₂; —N(R_(x))₂; —S(O)₂R_(x); —NR_(x)(CO)R_(x)wherein each occurrence of R_(x) independently includes, but is notlimited to, aliphatic, alicyclic, heteroaliphatic, heterocyclic,aromatic, heteroaromatic, aryl, heteroaryl, alkylaryl, alkylheteroaryl,heteroalkylaryl or heteroalkylheteroaryl, wherein any of the aliphatic,alicyclic, heteroaliphatic, heterocyclic, alkylaryl, or alkylheteroarylsubstituents described above and herein may be substituted orunsubstituted, branched or unbranched, saturated or unsaturated, andwherein any of the aromatic, heteroaromatic, aryl or heteroarylsubstituents described above and herein may be substituted orunsubstituted. Additional examples of generally applicable substituentsare illustrated by the specific embodiments shown in the Examples thatare described herein.

The term “heterocycloalkyl”, “heterocycle” or “heterocyclic”, as usedherein, refers to compounds which combine the properties ofheteroaliphatic and cyclic compounds and include, but are not limitedto, saturated and unsaturated mono- or polycyclic cyclic ring systemshaving 5-16 atoms wherein at least one ring atom is a heteroatomselected from S and N (wherein the nitrogen and sulfur heteroatoms maybe optionally be oxidized), wherein the ring systems are optionallysubstituted with one or more functional groups, as defined herein. Incertain embodiments, the term “heterocycloalkyl”, “heterocycle” or“heterocyclic” refers to a non-aromatic 5-, 6- or 7-membered ring or apolycyclic group wherein at least one ring atom heteroatom selected fromS and N (wherein the nitrogen and sulfur heteroatoms may be optionallybe oxidized), including, but not limited to, a bi- or tri-cyclic group,comprising fused six-membered rings having between one and threeheteroatoms independently selected from oxygen, sulfur and nitrogen,wherein (i) each 5-membered ring has 0 to 2 double bonds, each6-membered ring has 0 to 2 double bonds and each 7-membered ring has 0to 3 double bonds, (ii) the nitrogen and sulfur heteroatoms may beoptionally be oxidized, (iii) the nitrogen heteroatom may optionally bequaternized, and (iv) any of the above heterocyclic rings may be fusedto an aryl or heteroaryl ring. Representative heterocycles include, butare not limited to, heterocycles such as furanyl, pyranyl, pyrrolyl,thienyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl,imidazolidinyl, piperidinyl, piperazinyl, oxazolyl, oxazolidinyl,isooxazolyl, isoxazolidinyl, dioxazolyl, thiadiazolyl, oxadiazolyl,tetrazolyl, triazolyl, thiatriazolyl, thiadiazolyl, oxadiazolyl,morpholinyl, thiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl,dithiazolyl, dithiazolidinyl, tetrahydrofuryl, and benzofusedderivatives thereof. In certain embodiments, a “substituted heterocycle,or heterocycloalkyl or heterocyclic” group is utilized and as usedherein, refers to a heterocycle, or heterocycloalkyl or heterocyclicgroup, as defined above, substituted by the independent replacement ofone, two or three of the hydrogen atoms thereon with but are not limitedto aliphatic; alicyclic; heteroaliphatic; heterocyclic; aromatic;heteroaromatic; aryl; heteroaryl; alkylaryl; heteroalkylaryl;alkylheteroaryl; heteroalkylheteroaryl; alkoxy; aryloxy; heteroalkoxy;heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; F;Cl; Br; I; —OH; —NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH;—CH₂NH₂; —CH₂SO₂CH₃; —C(═O)R_(x); —C(═O)N(R_(x))₂, —OC(═O)R_(x);—OCO₂R_(x); —OC(═O)N(R_(x))₂; —N(R_(x))₂; —S(O)₂R_(x); —NR_(x)(CO)R_(x)wherein each occurrence of R_(x) independently includes, but is notlimited to, aliphatic, alicyclic, heteroaliphatic, heterocyclic,aromatic, heteroaromatic, aryl, heteroaryl, alkylaryl, alkylheteroaryl,heteroalkylaryl or heteroalkylheteroaryl, wherein any of the aliphatic,alicyclic, heteroaliphatic, heterocyclic, alkylaryl, or alkylheteroarylsubstituents described above and herein may be substituted orunsubstituted, branched or unbranched, saturated or unsaturated, andwherein any of the aromatic, heteroaromatic, aryl or heteroaryldescribed above and herein may be substituted or unsubstituted.Additionally, it will be appreciated that any of the alicyclic orheterocyclic moieties described above and herein may comprise an aryl orheteroaryl moiety fused thereto.

The terms “halo” and “halogen” used herein refer to an atom selectedfrom fluorine, chlorine, bromine and iodine.

The term “haloalkyl” denotes an alkyl group, as defined above, havingone, two, or three halogen atoms attached thereto and is exemplified bysuch groups as chloromethyl, bromoethyl, trifluoromethyl, and the like.

The term “amino” as used herein, refers to a primary (—NH₂), secondary(—NHR_(x)), tertiary (—NR_(x)R_(y)), or quaternary amine(—N⁺R_(x)R_(y)R_(z)), where R_(y) and R_(z) are independently analiphatic, alicyclic, heteroaliphatic, heterocyclic, aromatic orheteroaromatic moiety, as defined herein. Examples of amino groupsinclude, but are not limited to, methylamino, dimethylamino, ethylamino,diethylamino, diethylaminocarbonyl, iso-propylamino, piperidino,trimethylamino, and propylamino.

The term “acyl”, as used herein, refers to a group having the generalformula —C(═O)R, where R is an aliphatic, alicyclic, heteroaliphatic,heterocyclic, aromatic or heteroaromatic moiety, as defined herein.

The term “sulfonamido” as used herein, refers to a group of the generalformula —SO2NRxRy where Rx and Ry are independently hydrogen, or analiphatic, alicyclic, heteroaliphatic, heterocyclic, aromatic,heteroaromatic or acyl moiety, as defined herein.

The term “benzamido”, as used herein, refers to a group of the generalformula PhNRx, where Rx is hydrogen, or an aliphatic, alicyclic,heteroaliphatic, heterocyclic, aromatic, heteroaromatic or acyl moiety,as defined herein.

The term “C 1-6 alkylidene” as used herein, refers to a substituted orunsubstituted, linear or branched saturated divalent radical consistingsolely of carbon and hydrogen atoms, having from one to six carbonatoms, having a free valence “-” at both ends of the radical.

The term “C 2-6 alkylidene” as used herein, refers to a substituted orunsubstituted, linear or branched unsaturated divalent radicalconsisting solely of carbon and hydrogen atoms, having from two to sixcarbon atoms, having a free valence “-” at both ends of the radical, andwherein the unsaturation is present only as double bonds and wherein adouble bond can exist between the first carbon of the chain and the restof the molecule.

As used herein, the terms “aliphatic”, “heteroaliphatic”, “alkyl”,“alkenyl”, “alkynyl”, “heteroalkyl”, “heteroalkenyl”, “heteroalkynyl”,and the like encompass substituted and unsubstituted, saturated andunsaturated, and linear and branched groups. Similarly, the terms,“alicyclic”, “heterocyclic”, heterocycloalkyl”, “heterocycle” and thelike, encompass substituted and unsubstituted, and saturated andunsaturated groups. Additionally, the terms “cycloalkyl”, cycloalkenyl“,cycloalkynyl”, “heterocycloalkyl” “heterocycloalkenyl”,“heterocycloalkynyl”, “aromatic”, “heteroaromatic”, “aryl”, “heteroaryl”and the like encompass both substituted and unsubstituted groups.

The term “natural amino acid” as used herein refers to any one of thecommon, naturally occurring L-amino acids found in naturally occurringproteins: glycine (Gly), alanine (Ala), valine (Val), leucine (Leu),isoleucine (Ile), lysine (Lys), arginine (Arg), histidine (His), proline(Pro), serine (Ser), threonine (Thr), phenylalanine (Phe), tyrosine(Tyr), tryptophan (Trp), aspartic acid (Asp), glutamic acid (Glu),asparagine (Asn), glutamine (Gin), cysteine (Cys) and methionine (Met).

The term “unnatural amino acid” as used herein refers to all amino acidswhich are not natural amino acids. This includes, for example, α-, β-,D-, L-amino acid residues, and compounds of the general formula:

wherein the side chain R is other than the amino acid side chainsoccurring in nature.

More generally, the term “amino acid”, as used herein, encompassesnatural amino acids and unnatural amino acids.

The term “bioisosteres”, as used herein, generally refers to two or morecompounds or moieties that possess similar molecular shapes and/orvolumes. In certain embodiments, bioisosteres have approximately thesame distribution of electrons. In certain other embodiments,bioisosteres exhibit similar biological properties. In preferredembodiments, bioisosteres possess similar molecular shapes and volumes;have approximately the same distribution of electrons; and exhibitsimilar biological properties.

The term “pharmaceutically acceptable derivative”, as used herein,denotes any pharmaceutically acceptable salt, ester, or salt of suchester, of such compound, or any other adduct or derivative which, uponadministration to a patient, is capable of providing (directly orindirectly) a compound as otherwise described herein, or a metabolite orresidue thereof. Pharmaceutically acceptable derivatives thus includeamong others pro-drugs. A pro-drug is a derivative of a compound,usually with significantly reduced pharmacological activity, whichcontains an additional moiety, which is susceptible to removal in vivoyielding the parent molecule as the pharmacologically active species. Anexample of a pro-drug is an ester, which is cleaved in vivo to yield acompound of interest. Pro-drugs of a variety of compounds, and materialsand methods for derivatizing the parent compounds to create thepro-drugs, are known and may be adapted to the present invention.Certain exemplary pharmaceutical compositions and pharmaceuticallyacceptable derivatives will be discussed in more detail herein below.

As used herein, the term pharmaceutically acceptable salt” refers tothose salts which are suitable for pharmaceutical use, preferably foruse in the tissues of humans and lower animals without undue irritation,allergic response and the like. Pharmaceutically acceptable salts ofamines, carboxylic acids, and other types of compounds, are well knownin the art. For example, S. M. Berge, et al., describe pharmaceuticallyacceptable salts in detail in J Pharmaceutical Sciences, 66: 1-19(1977), incorporated herein by reference. The salts can be prepared insitu during the final isolation and purification of the compounds of theinvention, or separately by reacting a free base or free acid functionwith a suitable reagent, as described generally below. For example, afree base function can be reacted with a suitable acid. Furthermore,where the compounds of the invention carry an acidic moiety, suitablepharmaceutically acceptable salts thereof may, include metal salts suchas alkali metal salts, e. g. sodium or potassium salts; and alkalineearth metal salts, e. g. calcium or magnesium salts. Examples ofpharmaceutically acceptable, nontoxic acid addition salts are salts ofan amino group formed with inorganic acids such as hydrochloric acid,hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid orwith organic acids such as acetic acid, oxalic acid, maleic acid,tartaric acid, citric acid, succinic acid or malonic acid or by usingother methods used in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzoate, bisulfate, borate, butyrate, camphorate,camphorsulfonate, citrate, cyclopentanepropionate, digluconate,dodecylsulfate, formate, fumarate, glucoheptonate, glycerophosphate,gluconate, hernisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate, nicotinate,nitrate, oleate, oxalate, palmitate, pectinate, persulfate,3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate,succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate,undecanoate, valerate salts, and the like. Representative alkali oralkaline earth metal salts include sodium, lithium, potassium, calcium,magnesium, and the like. Further pharmaceutically acceptable saltsinclude, when appropriate, nontoxic ammonium, quaternary ammonium, andamine cations formed using counterions such as halide, hydroxide,carboxylate, sulfate, phosphate, nitrate, sulfonate and aryl sulfonate.

As used herein, the term “pharmaceutically acceptable ester” refers toesters that hydrolyze in vivo and include those that break down readilyin the human body to leave the parent compound or a salt thereof.Suitable ester groups include, for example, those derived frompharmaceutically acceptable aliphatic alcohol compounds, particularlyalkanes, alkenes, ehtylene glycol, cycloalkanes, and the like in whicheach alkyl or alkenyl moiety advantageously has not more than 6 carbonatoms. These are exemplary only and in no way limit the possibilities ofesters known in the art.

As used herein, the term “pharmaceutically acceptable prodrugs” refersto those prodrugs of the compounds of the present invention which aresuitable for pharmaceutical use, preferably for use with the tissues ofhumans and lower animals with undue toxicity, irritation, allergicresponse, and the like, and effective for their intended use, as well asthe zwitterionic forms, where possible, of the compounds of theinvention. The term “prodrug” refers to compounds that are rapidlytransformed in vivo to yield the parent compound of the above formula,for example by hydrolysis in blood. A thorough discussion is provided inT. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14of the A. C. S. Symposium Series, and in Edward B. Roche, ed.,Bioreversible Carriers in Drug Design, American PharmaceuticalAssociation and Pergamon Press, 1987, both of which are incorporatedherein by reference.

B. Exemplary Compounds of the Method.

In one embodiment, compounds useful in the methods of the presentinvention include compounds of Formula I:

where R¹ and R² are each independently hydrogen, an amino acid sidechain, —(CH₂)_(m)OH, —(CH2)_(m)aryl, —(CH2)_(m)heteroaryl, wherein m is0-6, —CH(R^(1A))(OR^(1B)), —CH(R^(1A))(NHR^(1B)), U-T-Q, or analiphatic, alicyclic, heteroaliphatic or heteroalicyclic moietyoptionally substituted with U-T-Q; wherein U may be absent or one of thefollowing: —O—, —S(O)₀₋₂—, —SO₂N(R^(1A)), —N(R^(1A))—, —N(R^(1A))C(═O)—,—N(R^(1A))C(═O)—O—, —N(R^(1A))C(═O)—N(R^(1B))—, —N(R^(1A))—SO₂—,—C(═O)—, —C(═O)—O—, —O—C(═O)—, aryl, heteroaryl, alkylaryl,alkylheteroaryl, —C(═O)—N(R^(1A))—, —OC(═O)N(R^(1A))—, —C(═N—R^(1E))—,—C(═N—R^(1E))—O—, —C(═N—R^(1E))—N(R^(1A))—, —O—C(═N—R^(1E))—N(R^(1A))—,—N(R^(1A))C(═N—R^(1E))—, —N(R^(1A))C(═N—R^(1E))—O—,—N(R^(1A))C(═N—R^(1E))—N(R^(1B))—, —P(═O)(OR^(1A))—O—, or—P(═O)(R^(1A))—O—; wherein T is absent or, an aliphatic,heteroaliphatic, aryl, heteroaryl, alkylaryl or alkylheteroaryl moiety;and Q is hydrogen, halogen, cyano, isocyanate, —OR^(1B); —SR^(1B);—N(R^(1B))₂, —NHC(═O)OR^(1B), —NHC(═O)N(R^(1B))₂, —NHC(═O)R^(1B),—NHSO₂R^(1B), NHSO₂N(R^(1B))₂, —NHSO₂NHC(═O)OR^(1B),—NHC(═O)NHSO₂R^(1B), —C(═O)NHC(═O)OR^(1B), C(═O)NHC(═O)R^(1B),—C(═O)NHC(═O) N(R^(1B))₂, —C(═O)NHSO₂R^(1B), —C(═O)NHSO₂N(R^(1B))₂,C(═S)N(R^(1B))₂, —SO₂R^(1B), —SO₂OR^(1B), —SO₂N(R^(1B))₂, —SO₂—NHC(═O)OR^(1B), —OC(═O)—N(R^(1B))2, —OC(═O)R^(1B), —OC(═O)NHC(═O)R^(1B),—OC(═O)NHSO₂R^(1B), —OSO₂R^(1B), or an aliphatic heteroaliphatic, arylor heteroaryl moiety, or wherein R¹ and R² taken together are analicyclic or heterocyclic moiety, or together are

wherein each occurrence of R^(1A) and R^(1B) is independently hydrogen,an aliphatic, alicyclic, heteroaliphatic, heterocyclic, aryl,heteroaryl, alkylaryl or alkylheteroaryl moiety, —C(═O)R^(1C), or—C(═O)NR^(1C)R^(1D); wherein each occurrence of R^(1C) and R^(1D) isindependently hydrogen, hydroxyl, or an aliphatic, heteroaliphatic,aryl, heteroaryl, alkylaryl or alkylheteroaryl moiety; and R^(1E) ishydrogen, an aliphatic, alicyclic, heteroaliphatic, heterocyclic, aryl,heteroaryl, alkylaryl or alkylheteroaryl moiety, —CN, —OR^(1C),—NR^(1C)R^(1D) or —SO2R^(1C);where R³ is —C(═O)OR^(3A), —C(═O)H, —CH₂OR^(3A), —CH₂OC(═O)-alkyl,—C(═O)NH(R^(3A)). —CH₂X⁰; wherein each occurrence of R^(1E) isindependently hydrogen, a protecting group, an aliphatic, alicyclic,heteroaliphatic, heteroalicyclic, aryl, heteroaryl, alkylaryl,alkylheteroaryl, heteroalkylaryl, heteroalkylheteroaryl moiety, or apharmaceutically acceptable salt or ester, or R^(3A), taken togetherwith R¹ and R², forms a heterocyclic moiety; wherein X⁰ is a halogenselected from F, Br or I;R⁴ for each occurrence, is independently hydrogen, halogen, —CN, —NO₂,an aliphatic, alicyclic, heteroaliphatic, heteroalicyclic, aryl,heteroaryl, alkylaryl or alkylheteroaryl moiety, or is GR^(G1) wherein Gis —O—, —S—, NR^(G2)—, —CO—, —SO—, —SO₂—, C(═O)O—, —C(═O)NR^(G2)—,C(═O)—, —NR^(G2)C(═O)— or —SO₂NR^(G2)—, and R^(G1) and R^(G2) areindependently hydrogen, an aliphatic, alicyclic, heteroaliphatic,heteroalicyclic, aryl, heteroaryl, alkylaryl or alkylheteroaryl moiety;n is an integer from 0-4;AR¹ is a monocyclic or polycyclic aryl, heteroaryl, alkylaryl,alkylheteroaryl, alicyclic or heterocyclic moiety;A, B, D and E are connected by either a single or double bond, asvalency permits; wherein each occurrence of A, D and E is independentlyC═O, CR^(i)R^(ii), NR^(i), CR^(i), N, O, S, —S(═O) or SO₂; wherein eachoccurrence of R^(i) is independently hydrogen, halogen, —CN, —NO2, analiphatic, alicyclic, heteroaliphatic, heteroalicyclic, aryl,heteroaryl, alkylaryl or alkylheteroaryl moiety, or is —GR^(G1) whereinG is —O—, —S—, —NR^(G2), —CO—, —SO—, —C(═O)O—, —C(═O) NR^(G2)—,—OC(═O)—, —NR^(G2)C(═O)— or —SO₂NR^(G2), and R^(G1) and R^(G2) areindependently hydrogen, an aliphatic, alicyclic, heteroaliphatic,heteroalicyclic, aryl, heteroaryl, alkylaryl or alkylheteroaryl moiety,or any two adjacent occurrences of taken together, represent analicyclic, heteroalicyclic, aryl, or heteroaryl moiety;p is an integer from 0-4; and,L is absent or is V—W—X—Y—Z, wherein each occurrence of V, W, X, Y and Zis independently absent, C═O, NR^(L1), —C(R^(L1))═, ═C(R^(L1))—,—C(R^(L1))(R^(L2)), C(═N—OR^(L1)), C(═NR^(L1)), —N═, S(O)₀₋₂; asubstituted or unsubstituted C₁₋₆ alkenylidene or C₂₋₆ alkenylidinechain wherein up to two non-adjacent methylene units are independentlyoptionally replaced by —C(═O)—, —CO₂—, —C(═O)C(═O)—, —C(C═O)NR^(L3)—,—OC(═O)—, —OC(═O)NR^(L3)—, —NR^(L3)NR^(L4)—, —NR^(L3)NR^(L4)C(═O)—,—NR^(L3)C(═O)—, NR^(L3)CO₂—, NR^(L3)C(═O)NR^(L4)—, —S(═O)—, —SO₂—,—NR^(L3)SO₂ ⁻, —SO₂NR^(L3), —NR^(L3)SO₂NR^(L4), —O—, —S—, or —NR^(L3)—;wherein each occurrence of R^(L3) and R^(L4) is independently hydrogen,alkyl, heteroalkyl, aryl, heteroaryl or acyl; or an aliphatic,alicyclic, heteroaliphatic, heteroalicyclic, aryl, heteroaryl, alkylarylor alkylheteroaryl moiety; and each occurrence of R^(L1) and R^(L2) isindependently hydrogen, hydroxyl, protected hydroxyl, amino, protectedamino, thio, protected thio, halogen, cyano, isocyanate, carboxy,carboxyalkyl, formyl, formyloxy, azido, nitro, ureido, thioureido,thiocyanato, alkoxy, aryloxy, mercapto, sulfonamido, benzamido, tosyl,or an aliphatic, alicyclic, heteroaliphatic, heteroalicyclic, aryl,heteroaryl, alkylaryl or alkylheteroaryl moiety, or wherein one or moreoccurrences of R^(L1) and R^(L2), taken together, or taken together withone of V, W, X, Y or Z form an alicyclic or heterocyclic moiety or forman aryl or heteroaryl moiety.

Some preferred embodiments of the method of the present invention are ofFormula II:

where R²⁸ is one of the following groups:

And R²⁷ is one of the following groups:

and R²⁹ is hydrogen, a pharmaceutically acceptable salt or ester.

Some preferred embodiments of the invention are compounds of the FormulaII′

where the substitution is as in Formula II.

Some particularly preferred embodiments of compounds of the method ofthe present invention are compounds of Formulas IIA, IIB and IIC:

where R¹⁷ respectively can be chosen from the group of hydrogen,pharmaceutically acceptable salts and esters.

Another set of preferred embodiments of compounds of the method of theinvention are compounds of the Formula III:

where Cy is an aromatic carbocycle, aromatic heterocycle or anon-aromatic carbocycle or heterocycle optionally substituted withhydroxyl (—OH), mercapto (—SH), thioalkyl, halogen (e.g. F, Cl, Br, I),oxo (═O), thio (═S), amino, aminoalkyl, amidine (—C(NH)—NH₂), guanidine(—NH₂—C(NH)—NH₂), nitro, alkyl or alkoxy. In a particular embodiment, Cyis a 3-5 member ring. In a preferred embodiment, Cy is a 5- or 6-membernon-aromatic heterocycle optionally substituted with hydroxyl, mercapto,halogen (preferably F or Cl), oxo (═O), thio (═S), amino, amidine,guanidine, nitro, alkyl or alkoxy. In a more preferred embodiment, Cy isa 5-member non-aromatic heterocycle optionally substituted withhydroxyl, oxo, thio, Cl, C₁₋₄ alkyl (preferably methyl), or C₁₋₄alkanoyl (preferably acetyl, propanoyl or butanoyl). More preferably thenon-aromatic heterocycle comprises one or heteroatoms (N, O or S) and isoptionally substituted with hydroxyl, oxo, mercapto, thio, methyl,acetyl, propanoyl or butyl. In particular embodiments the non-aromaticheterocycle comprises at least one nitrogen atom that is optionallysubstituted with methyl or acetyl. In a particularly preferredembodiment, the non-aromatic heterocycle is selected from the groupconsisting of piperidine, piperazine, morpholine, tetrahydrofuran,tetrahydrothiophene, oxazolidine, thiazolidine optionally substitutedwith hydroxy, oxo, mercapto, thio, alkyl or alkanoyl. In a mostpreferred embodiment Cy is a non-aromatic heterocycle selected from thegroup consisting of tetrahydrofuran-2-yl, thiazolidin-5-yl,thiazolidin-2-one-5-yl, and thiazolidin-2-thione-5-yl andcyclopropapyrrolidine. In a preferred embodiment, Cy is a 5- or 6-memberaromatic carbocycle or heterocycle optionally substituted with hydroxyl,mercapto, halogen (preferably F or Cl), oxo (═O), thio (═S), amino,amidine, guanidine, nitro, alkyl or alkoxy. In a more preferredembodiment, Cy is a 5-member aromatic carbocycle or heterocycleoptionally substituted with hydroxyl, oxo, thio, Cl, C₁₋₄ alkyl(preferably methyl), or C₁₋₄ alkanoyl (preferably acetyl, propanoyl orbutanoyl). More preferably the aromatic or heterocycle comprises one orheteroatoms (N, O or S) and is optionally substituted with hydroxyl,oxo, mercapto, thio, methyl, acetyl, propanoyl or butyl.

In another preferred embodiment Cy is a 3-6 member carbocycle optionallysubstituted with hydroxyl, mercapto, halogen, oxo, thio, amino, amidine,guanidine, alkyl, alkoxy or acyl. In a particular embodiment thecarbocycle is saturated or partially unsaturated. In particularembodiments Cy is a carbocycle selected from the group consisting ofcyclopropyl, cyclopropenyl, cyclobutyl, cyclobutenyl, cyclopentyl,cyclopentenyl, cyclohexyl and cyclohexenyl. X₂ is a C₁₋₅ divalenthydrocarbon linker optionally having one or more carbon atoms replacedwith N, O, S, SO or SO₂ and optionally being substituted with hydroxyl,mercapto, halogen, amino, aminoalkyl, nitro, oxo or thio. In a preferredembodiment X₂ will have at least one carbon atom. Replacements andsubstitutions may form an amide moiety (—NRC(═O)— or —C(═O)NR—) withinthe hydrocarbon chain or at either or both ends. Other moieties includesulfonamide (—NRSO₂— or —SO₂NR), acyl, ether, thioether and amine. In aparticularly preferred embodiment X₂ is the group —CH₂—NR¹⁰—C(O)—wherein the carbonyl —C(O)— portion thereof is adjacent (i.e. covalentlybound) to Cy and R¹⁰ is alkyl i.e. methyl and more preferably H.

K is a carbocycle or heterocycle optionally substituted with hydroxyl,mercapto, halogen, oxo, thio, a hydrocarbon, a halo-substitutedhydrocarbon, amino, amidine, guanidine, cyano, nitro, alkoxy or acyl. Inparticular embodiment, K is aryl or heteroaryl optionally substitutedwith halogen or hydroxyl. In a particularly preferred embodiment, K isphenyl, furan-2-yl, thiophene-2-yl, phenyl substituted with a halogen(preferably Cl) or hydroxyl, preferably at the meta position. L₂ is adivalent hydrocarbon optionally having one or more carbon atoms replacedwith N, O, S, SO or SO₂ and optionally being substituted with hydroxyl,halogen oxo, or thio; or three carbon atoms of the hydrocarbon arereplaced with an amino acid residue. Preferably L₂ is less than 10 atomsin length and more preferably 5 or less and most preferably 5 or 3 atomsin length. In particular embodiments, L₂ is selected from the groupconsisting of —CH═CH—C(O)—NR¹⁰—CH₂—, —CH₂—NR¹⁰—C(O)—, —C(O)—NR¹⁰—CH₂—,—CH(OH)—(CH₂)₂—, —(CH₂)₂—CH(OH)—, —(CH₂)₃—, —C(O)—NR¹⁰—CH(R₇)—C(O)—NR¹⁰—, —NR¹⁰—C(O)—CH(R¹⁶)—NR¹⁰—C(O)—, —CH(OH)—CH₂—O— and—CH(OH)—CF₂—CH₂— wherein each R¹⁰ is independently H or alkyl and R¹⁶ isan amino acid side chain. Preferred amino acid side chains includenon-naturally occurring side chains such as phenyl or naturallyoccurring side chains Preferred side chains are those from Phe, Tyr,Ala, Gln and Asn. In a preferred embodiments L₂ is —CH═CH—C(O)—NR¹⁰—CH₂—wherein the —CH═CH— moiety thereof is adjacent (i.e. covalently bound)to K. In another preferred embodiment, L₂ is —CH₂—NR¹⁰—C(O)— wherein themethylene moiety (—CH₂—) thereof is adjacent to K.

R⁵ is H, OH, amino, O-carbocycle or alkoxy optionally substituted withamino, a carbocycle, a heterocycle, or a pharmaceutically acceptablesalt or ester. In a preferred embodiment, R⁵ is H, phenyl or C₁₋₄ alkoxyoptionally substituted with a carbocycle such as phenyl. In a particularembodiment R⁵ is H. In another particular embodiment R⁵ is methoxy,ethoxy, propyloxy, butyloxy, isobutyloxy, s-butyloxy, t-butyloxy,phenoxy or benzyloxy. In yet another particular embodiment R⁵ is NH₂. Ina particularly preferred embodiment R⁵ is ethoxy. In anotherparticularly preferred embodiment R⁵ is isobutyloxy. In anotherparticularly preferred embodiment R⁵ is alkoxy substituted with amino,for example 2-aminoethoxy, N-morpholinoethoxy, N,N-dialkyaminoethoxy,quaternary ammonium hydroxy alkoxy (e.g.trimethylammoniumhydroxyethoxy).R⁶⁻⁹ are independently H, hydroxyl, mercapto, halogen, cyano, amino,amidine, guanidine, nitro or alkoxy; or R⁷ and R⁸ together form a fusedcarbocycle or heterocycle optionally substituted with hydroxyl, halogen,oxo, thio, amino, amidine, guanidine or alkoxy. In a particularembodiment R⁶ and R⁷ are independently H, F, Cl, Br or I. In anotherparticular embodiment, R⁸ and R⁹ are both H. In another particularembodiment, one of R⁶ and R⁷ is a halogen while the other is hydrogen ora halogen. In a particularly preferred embodiment, R⁷ is Cl while R⁶, R⁸and R⁹ are each H. In another particularly preferred embodiment, R⁶ andR⁷ are both Cl while R⁸ and R⁹ are both H.R¹⁰ is H or a hydrocarbon chain optionally substituted with a carbocycleor a heterocycle. In a preferred embodiment, R¹⁰ is H or alkyl i.e.methyl, ethyl, propyl, butyl, i-butyl, s-butyl or t-butyl. In aparticular embodiment R¹⁰ is H.

Further preferred embodiments of the method of the present invention arecompounds of the Formula IV:

where R¹¹ is a group of the formula:

where A is hydrogen, hydroxy, amino, or halogen and B is amino, carboxy,hydrogen, hydroxy, cyano, trifluoromethyl, halogen, lower alkyl, orlower alkoxy;R¹² is a group of the formula:

where R¹³ is hydrogen, carboxy, or lower alkyl; n is 0 or 1; U₂, V₂, andW₂ are independently hydrogen, halogen, or lower alkyl provided U₂ andV₂ are not both hydrogen; X₃ is carbonyl, phenyl-substituted loweralkylene, imino, substituted imino, or sulfonyl; Y₂ is lower alkylenewhich may be substituted by one or more of amino, substituted amino,lower alkyl, or cyclo lower alkyl, or Y₂ is lower alkenylene or loweralkylenethio;k is 0 or 1; when k is 1, Z₂ is hydrogen, lower alkylthio, —COOH,—CONH₂, amino; and when k is 0 or 1, Z₂ is 1-adamantyl, diphenylmethyl,3-[[(5-chloropyridin-2-yl) amino] carbonyl] pyrazin-2-yl, hydroxy,phenylmethoxy, 2-chloro-4-[[[(3-hydroxyphenyl) methyl] amino] carbonyl]phenyl, [2,6-dichlorophenyl) methoxy] phenyl; further when k is 0 or 1,Z₂ may be cycloalkyl or aryl containing 0 to 3 heteroatoms which may bethe same or different, or a fused ring system containing two or threerings which rings are independently cycloalkyl or aryl containing 0 to 3heteroatoms which may be the same or different, any of which rings maybe unsubstituted, or substituted with at least one of halogen, cyano,amino, substituted amino, aminosulfonyl, nitro, oxo, hydroxy, aryl,aryloxy, unsubstituted lower alkyl, halogen-substituted lower alkyl,lower alkoxy-substituted lower alkyl, lower alkoxy, loweralkanesulfonyl, lower alkylthio, acetyl, aminocarbonyl, hydrazino,carboxy, alkoxycarbonyl, acetoxy, or also in addition with amino loweralkyl; and R²⁰ is hydrogen, a pharmaceutically acceptable salt or ester.

A preferred embodiment of compounds of Formula IV has stereochemistry asindicated in Formula IV:

Another set of preferred embodiments of the compounds of the method ofthe present invention are compounds of Formula V:

where R¹⁴ is a group of the formula:

where R¹⁵ is hydrogen, carboxy, or lower alkyl; U₃, V₃, and W₃ areindependently hydrogen, halogen; or U₃, V₃, and W₃ are lower alkylprovided that U₃ and V₃ are not both hydrogen; X₄ is carbonyl,phenyl-substituted lower alkylene, imino, substituted imino whichincludes cyano, or sulfonyl; Y₃ is lower alkenylene, lower alkylenethio,or is lower alkylene which may be substituted by amino, acetylamino, orcyclo-lower alkyl;k₂ is 0 or 1; when k₂ is 1, Z is hydrogen, lower alkylthio, —COOH,—CONH₂—, or amino; when k₂ is 0 or 1, Z₃ is 1-adamantyl, diphenylmethyl,3-[[(5-chloropyridin-2-yl)amino]carbonyl]pyrazin-2-yl; and when k₂ is 0or 1, Z may be cycloalkyl or aryl containing 0 to 3 heteroatoms whichmay be the same or different, or a fused ring system containing two orthree rings which rings are independently cycloalkyl or aryl containing0 to 3 heteroatoms which may be the same or different, any of whichrings may be unsubstituted, or substituted with at least one of halogen,cyano, amino, substituted amino, aminosulfonyl, nitro, oxo, hydroxy,aryl, aryloxy, unsubstituted lower alkyl, halogen-substituted loweralkyl, lower alkoxy-substituted lower alkyl, lower alkoxy, carboxy,alkoxycarbonyl, or acetoxy; and, R²¹ is hydrogen, pharmaceuticallyacceptable salts or esters thereof.

A preferred embodiment of compounds of Formula V has the stereochemistryas indicated in Formula V′:

Another class of preferred compounds of the method are represented byFormula VI

where D₄ is a mono-, bi-, or tricyclic saturated, unsaturated, oraromatic ring, each ring having 5-, 6- or 7 atoms in the ring where theatoms in the ring are carbon or from one to four heteroatoms selectedfrom the group nitrogen, oxygen, and sulfur, where any carbon or sulfurring atom may optionally be oxidized, each ring substituted with 0-3R³¹;L₃ is a bivalent linking group selected from the group-L³-L²-L¹-,-L⁴-L³-L²-L,¹- and-L⁵-L⁴-L³-L²-L¹-,where L¹ is selected from oxo (—O—), S(O)_(s), C(═O), CR³², R³², CR³²het, NR³⁰ and N,L² is selected from oxo (—O—), S(O)_(s), C(═O), C(═N—O—R³³),CR³⁴R³⁴′, CR³⁴, het NR³⁰ and N,L³ is selected from oxo (—O—), S(O)_(s), C(═O), C(═N—O—R³³), CR³⁵R³⁵′,CR³⁵, het NR³⁰ and N,L⁴ is absent or is selected from oxo (—O—), S(O)_(s), C(═O),C(═N—O—R³³), CR³⁶R³⁶′, CR³⁶, NR³⁰ and N,L⁵ is absent or selected from oxo (—O—), S(O)_(s), C(═O), CR³⁷R³⁷′,CR³⁷, NR³⁰ and N, provided that only one of L¹-L³ may be het and thatwhen one of L¹-L³ is het the other L¹-L⁵ may be absent,whereR³², R³²′, R³⁴, R³⁴′, R³⁵, R³⁵′, R³⁶, R³⁶, R³⁷ and R^(37′) each areindependently selected from R³⁸, R³⁹ and

U-Q-V-W,

optionally, R²⁴ and R³⁴′ separately or together may form a saturated,unsaturated or aromatic fused ring with B₃ through a substituent RP onB, the fused ring containing 5, 6 or 7 atoms in the ring and optionallycontaining 1-3 heteroatoms selected from the group O, S and N, where anyS or N may optionally be oxidized;optionally, R³⁵ and R³⁵ separately or together and R³⁶ and R³⁶′separately or together may form a saturated, unsaturated or aromaticfused ring with D₃ through a substituent R³¹ on D₃, the fused ringcontaining 5, 6 or 7 atoms in the ring and optionally containing 1-3heteroatoms selected from the group O, S and N, where any S or N mayoptionally be oxidized; also optionally, each R³²—R³⁷, NR³⁰ or N inL¹-L⁵ together with any other R³²—R³⁷, NR³⁰ or N in L¹-L⁵ may form a 5,6 or 7 member homo- or heterocycle either saturated, unsaturated oraromatic optionally containing 1-3 additional heteroatoms selected fromN, O and S, where any carbon or sulfur ring atom may optionally beoxidized, each cycle substituted with 0-3 R³¹; and where s is 0-2; B isselected from the group

is a fused hetero- or homocyclic ring containing 5, 6 or 7 atoms, thering being unsaturated, partially saturated or aromatic, the heteroatomsselected from 1-3 O, S and N,Y₃ is selected from CH and NR³⁰, n is 0-3:G₃ is selected from hydrogen and C₁-C₆alkyl, optionally G taken togetherwith T may form a C₃-C₆cycloalkyl optionally substituted with —V—W;T₃ is selected from the groupa naturally occurring α-amino-acid side chain,and U₄-Q₄-V₄—W₄;U₄ is an optionally substituted bivalent radical selected from the groupC₁-C₆alkyl, C₀-C₆alkyl-Q, C₂-C₆alkenyl-Q, and C₂-C₆alkynyl-Q:where the substituents on any alkyl, alkenyl or alkynyl are 1-3 R³⁸;Q₄ is absent or is selected from the group—O—, —S(O)_(s)—, —SO₂—N(R³⁰)—, —N(R³⁰)—, —N(R³⁰)—C(═O)—,—N(R³⁰)—C(═O)—N(R³⁰)—, —N(R³⁰)—C(═O)—O—, —N(R³⁰)—SO₂—, —C(═O)—,—C(═O)—O—, -het-, —C(═O)—N(R³⁰)—, —O—C(═O)—N(R³⁰)—, —PO(OR³⁰)O— and—P(O)O—;wheres is 0-2 andhet is a mono- or bicyclic 5, 6, 7, 9 or 10 member heterocyclic ring,each ring containing 1-4 heteroatoms selected from N, O and S, where theheterocyclic ring may be saturated, partially saturated, or aromatic andany N or S being optionally oxidized, the heterocyclic ring beingsubstituted with 0-3 R⁴¹;V₄ is absent or is an optionally substituted bivalent group selectedfrom C₁-C₆alkyl, C₃-C₈cycloalkyl, C₀-C₆alkyl-C₆-C₁₀aryl, andC₀-C₆alky-het;where the substituents on any alkyl are 1-3 R³⁸ and the substituents onany aryl or het are 1-3 R³¹;W₄ is selected from the grouphydrogen, OR³³, SR⁴², NR³⁰R³⁰, NH—C(═O)—O—R⁴³, NH—C(═O)—NR^(n)R^(n),NH—C(═O)—R⁴³, NH—SO₂—R³⁷, NH—SO₂—NR³⁰R³⁰, NH—SO₂—NH—C(═O)—R⁴³,NH—C(═O)—NH—SO₂—R³⁷, C(═O)—NH—C(═O)—O—R⁴³, C(═O)—NH—C(═O)R⁴³,C(═O)—NH—C(═O)—NR³⁰R³⁰′, C(═O)—NH—SO₂—R³⁷, C(═O)—NH—SO₂—NR³⁰R³⁰′,C(═S)—NR³⁰R³⁰′, SO₂—R³⁷, SO₂—O—R³⁷, SO₂—NR³⁷R³⁷′, SO₂—NH—C(═O)—O—R⁴³,SO₂—NH—C(═O)—NR³⁰R³⁰′, SO₂—NH—C(═O)—R⁴³, O—C(═O)—NR³⁰R³⁰′, O—C(═O)—R⁴³,O—C(═O)—NH—C(═O)—R⁴³, O—C(═O)—NH—SO₂R⁴⁶ and O—SO₂—R³⁷;R⁴⁴ is selected from C(═O)—R⁴⁵, C(═O)—H, CH₂(OH), andCH₂O—C(═O)—C₁-C₆alkyl;R³⁸ is R³⁸′ or R³⁸″ substituted with 1-3 R³⁸′; whereR³⁸′ is selected from the grouphydrogen, halo(F. Cl, Br, I), cyano, isocyanate, carboxy,carboxy-C₁-C₁₁alkyl, amino, amino-C₁-C₈alkyl, aminocarbonyl,carboxamido, carbamoyl, carbamoyloxy, formyl, formyloxy, azido, nitro,imidazoyl, ureido, thioureido, thiocyanato, hydroxy, C₁-C₆alkoxy,mercapto, sulfonamido, het, phenoxy, phenyl, benzamido, tosyl,morpholino, morpholinyl, piperazinyl, piperidinyl, pyrrolinyl,imidazolyl, and indolyl;R³⁸″ is selected from the groupC₀-C₁₀alkyl-Q-C₀-C₆alkyl, C₀-C₁₀alkenyl-Q-C₀-C₆alkyl,C₀-C₁₀alkynyl-Q-C₀-C₆alkyl, C₃-C₁₁cycloalkyl-Q-C₀-C₆alkyl,C₃-C₁₀cycloalkenyl-Q-C₀-C₆alkyl, C₁-C₆alkyl-C₆-C₁₂ aryl-Q-C₀-C₆alkyl,C₆-C₁₀ aryl-C₁-C₆alkyl-Q-C₀-C₆alkyl, C₀- C₆alkyl-het-Q-C₀-C₆alkyl,C₀-C₆alkyl-Q-het-C₀-C₆alkyl, het-C₀-C₆alkyl-Q-C₀-C₆alkyl,C₀-C₆alkyl-Q-C₆-C₁₂aryl, and -Q-C₁-C₆alky;R⁴³ is selected from hydrogen and substituted or unsubstitutedC₁-C₁₀alkyl, C₂-C₁₀alkenyl, C₂-C₁₀alkynyl, C₃-C₁₁cycloalkyl,C₃-C₁₀cycloalkenyl, C₁-C₆alkyl-C₆-C₁₂aryl, C₆-C₁₀aryl-C₁-C₆alkyl,C₁-C₆alkyl-het, het-C₁-C₆ alkyl, C₆-C₁₂aryl and het,where the substituents on any alkyl, alkenyl or alkynyl are 1-3 R³⁸ andthe substituents on any aryl or het are 1-3 R³¹;R³¹ is selected from R⁴⁰ and R⁴¹;R⁴¹ is selected from the groupOH, OCF₃, OR⁴³, SR⁴², halo(F, Cl. Br, I), CN, isocyanate, NO₂, CF₃,C₀-C₆alkyl-NR³⁰R³⁰′, C₀-C₆alkyl-C(═O)—NR³⁰R³⁰′, C₀-C₆alkyl-C(═O)—R³⁸,C₁-C₈alkyl, C₁-C₈alkoxy, C₂-C₈alkenyl, C₂-C₈alkynyl, C₃-C₆cycloalkyl,C₃-C₆cycloalkenyl, C₁-C₆alkyl-phenyl, phenyl-C₁-C₆alkyl,C₁-C₆alkyloxycarbonyl, phenyl-C₀-C₆alkyloxy, C₁-C₆alkyl-het,het-C₁-C₆alkyl, SO₂-het, —O—C₆-C₁₂aryl, —SO₂—C₆-C₁₂aryl, —SO₂—C₁-C₆alkyland het,where any alkyl, alkenyl or alkynyl may optionally be substituted with1-3 groups selected from OH, halo(F, Cl, Br, I), nitro, amino andaminocarbonyl and the substituents on any aryl or het are 1-2 hydroxy,halo(F, Cl, Br, I), CF₃, C₁-C₆alkyl, C₁-C₆alkoxy, nitro and amino;R⁴² is selected from S—C₁-C₆alkyl, C(═O)—C₁-C₆alkyl, C(═O)—NR³⁰R³⁰′,C₁-C₆alkyl, halo(F, Cl, Br, I)—C₁-C₆alkyl, benzyl and phenyl;R³⁰ is selected from the group R⁴³, NH—C(═O)—O—R⁴³, NH—C(═O)—R⁴³,NH—C(═O)—NHR⁴³, NH—SO₂—R⁴⁶, NH—SO₂—NH—C(═O)—R⁴³, NH—C(═O)—NH—SO₂—R³⁷,C(═O)—O—R⁴³, C(═O)—R⁴³, C(═O)—NHR⁴³, C(═O)—NH—C(═O)—O—R⁴³,C(═O)—NH—C(═O)—R⁴³, C(═O)—NH—SO₂—R⁴⁶, C(═O)—NH—SO₂—NHR³⁷, SO₂—R³⁷,SO₂—O—R³⁷, SO₂—N(R⁴³)₂, SO₂—NH—C(═O)—O—R⁴³, SO₂—NH—C(═O)—O—R⁴³ andSO₂—NH—C(═O)R⁴³;R³⁰′ is selected from hydrogen, hydroxy and substituted or unsubstitutedC₁-C₁₁alkyl, C₁-C₁₁ alkoxy, C₂-C₁₀alkenyl, C₂-C₁₀alkynyl,C₃-C₁₁cycloalkyl, C₃-C₁₀cycloalkenyl, C₁-C₆alkyl-C₆-C₁₂aryl,C₆-C₁₀aryl-C₁-C-₆ alkyl, C₆-C₁₀aryl-C₀-C₆alkyloxy, C₁-C₆alkyl-het,het-C₁-C₆alkyl, C₆-C₁₂aryl, het, C₁-C₆alkylcarbonyl,C₁-C₈alkoxycarbonyl, C₃-C₈cycloalkylcarbonyl, C₃-C₈cycloalkoxycarbonyl,C₆-C₁₁aryloxycarbonyl, C₇-C₁₁arylalkoxycarbonyl,heteroarylalkoxycarbonyl, heteroarylalkylcarbonyl, heteroarylcarbonyl,heteroarylalkylsulfonyl, heteroarylsulfonyl, C₁-C₆alkylsulfonyl, andC₆-C₁₀arylsulfonyl, where the substituents on any alkyl, alkenyl oralkynyl are 1-3 R³⁸ and the substituents on any aryl, het or heteroarylare 1-3 R³¹;R³⁰ and R³⁰′ taken together with the common nitrogen to which they areattached may from an optionally substituted heterocycle selected frommorpholinyl, piperazinyl, thiamorpholinyl, pyrrolidinyl, imidazolidinyl,indolinyl, isoindolinyl, 1,2,3,4-tetrahydro-quinolinyl,1,2,3,4-tetrahydro-isoquinolinyl, thiazolidinyl and azabicyclononyl,where the substituents are 1-3 R³⁸;R³³ is selected from hydrogen and substituted or unsubstitutedC₁-C₆alkyl, C₁-C₆alkylcarbonyl, C₂-C₆alkenyl, C₂-C₆alkynyl,C₃-C₈cycloalkyl and benzoyl, where the substituents on any alkyl are 1-3R³⁸ and the substituents on any aryl are 1-3 R⁴⁰;R⁴⁰ is selected from the group OH, halo(F, Cl. Br, I), CN, isocyanate,OR⁴³, SR⁴², SOR⁴³, NO₂, CF₃, R⁴³, NR³⁰R³⁰′, NR³⁰C(═O)—O—R⁴³,NRC(═O)—R⁴³, C₀-C₆alkyl-SO₂—R⁴³, C₀-C₆alkyl-SO₂—NR³⁰R³⁰′, C(═O)—R⁴³,O—C(═O)R⁴³, C(═O)—O—R⁴³, and C(═O)—NR³⁰R³⁰′, where the substituents onany alkyl, alkenyl or alkynyl are 1-3 R³⁸ and the substituents on anyaryl or het are 1-3 R³¹;R⁴⁶ is a substituted or unsubstituted group selected fromC₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl, C₃-C₈cycloalkyl,C₃-C₆cycloalkenyl, C₀-C₆alkyl-phenyl, phenyl-C₀-C₆alkyl, C₀-C₆alkyl-hetand het-C₀-C₆alkyl,where the substituents on any alkyl, alkenyl or alkynyl are 1-3 R³⁸ andthe substituents on any aryl or het are 1-3 R³¹;R⁴⁵ is a substituted or unsubstituted group selected from hydroxy,C₁-C₁₁alkoxy, C₃-C₁₂cycloalkoxy, C₈-C₁₂aralkoxy, C₈-C₁₂arcycloalkoxy,C₆-C₁₀aryloxy, C₃-C₁₀ alkylcarbonyloxyalkyloxy, C₃-C₁₀alkoxycarbonyloxyalkyloxy, C₃-C₁₀alkoxycarbonylalkyloxy, C₅-C₁₀cycloalkylcarbonyloxyalkyloxy, C₅-C₁₀cycloalkoxycarbonyloxyalkyloxy,C₅-C₁₀cycloalkoxycarbonylalkyloxy, C₈-C₁₂aryloxycarbonylalkyloxy,C₈-C₁₂aryloxycarbonyloxyalkyloxy, C₈-C₁₂arylcarbonyloxyalkyloxy,C₅-C₁₀alkoxyalkylcarbonyloxyalkyloxy, (R³⁰)(R³⁰)N(C₁-C₁₀alkoxy)-,

where the substituents on any alkyl, alkenyl or alkynyl are 1-3 R³⁸ andthe substituents on any aryl or het are 1-3 R³¹ and pharmaceuticallyacceptable salts thereof.Compounds of Formulas I-VI also include pharmaceutically acceptablesalts, and esters including pro-drug compounds of Formula I-VI, whereR^(3A), R⁵, R¹⁰, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²⁹, and a carboxylic ester atR⁴⁴ may be lower alkyl or —CH₂CH₂—R²² where R²² is one of the following:

where R²³ is hydrogen or methyl and R²⁴ is lower alkyl or lowercycloalkyl.

A preferred embodiment of compounds of Formula VI has thestereochemistry indicated in Formula VI′.

Some of the compounds described herein may comprise one or moreasymmetric centers, and thus may comprise individual stereoisomers,individual diastereomers and any mixtures therein. Further, compounds ofthe invention may contain geometric isomers of double bonds, comprisingZ and E isomers, and may be present as pure geometric isomers ormixtures thereof.

In some preferred embodiments, the methods of the present invention areperformed with the following compounds or a pharmaceutically acceptablesalt or ester thereof:

Compounds of the present invention include the following compounds or apharmaceutically acceptable salt or ester thereof:

The compounds of the invention may be prepared by methods well known tothose skilled in the art and may be purified in a number of ways,including by crystallization or precipitation under varied conditions toyield one or more polymorphs. Thus, the present invention encompassesthe above described inventive compounds, their polymorphs, theirpharmaceutically acceptable salts, their pharmaceutically acceptablesolvates, and pharmaceutically acceptable compositions containing them.

The above examples of preferred embodiments are meant to illustrate someof the potential therapeutic agents, and are not meant to limit theinvention in any way. The method of the invention can be practiced withantibodies, fragments of antibodies, peptides and other syntheticmolecules that can be identified using the methods described above toidentify a therapeutic agent that is a selective, potent and directlycompetitive inhibitor of the interaction between LFA-1 and ICAM-1, inorder to treat dry eye disease.

Also provided herein are business methods which employ the compounds anddiagnostic and therapeutic methods described herein. One business methodinvolves the identification of LFA-1 antagonistic properties of peptidesor small molecules and developing the compounds for treatment of LFA-1mediated diseases, preferably by topical delivery. As the compounds arenot administered systemically, the systemic pharmacokinetic profiles ofthese drugs are typically not determined and hence the candidate pool ofdrugs available for development is larger. In one embodiment, the LFA-1antagonists are developed into ocular formulations and then promoted andsold for treatment of eye disorders, such as dry eye. The Hut78 assay istypically used to determine LFA-1 antagonistic properties. In additionto LFA-1 antagonistic properties, leukocyte antagonistic properties canbe determined.

III. Administration

The method of the present invention may draw upon many suitable modes ofadministration to deliver the LFA-1 antagonist of the methods describedherein. Such delivery to affected regions of the body may be achievedeither via local or systemic administration. Suitable formulations andadditional carriers are described in Remington “The Science and Practiceof Pharmacy” (20^(th) Ed., Lippincott Williams & Wilkins, BaltimoreMd.), the teachings of which are incorporated by reference in theirentirety herein.

In some embodiments, the invention provides a pharmaceutical compositionfor administration to a subject containing: (i) an effective amount of atherapeutic agent; and (ii) a pharmaceutical excipient suitable for oraladministration. In some embodiments, the composition further contains:(iii) an effective amount of a second therapeutic agent.

In order to reduce inflammation in eye disorders, the pharmaceuticalcomposition of the invention is preferably delivered to the ocularsurface, interconnecting innervation, conjuncitva, lacrimal glands, ormeibomian glands. It is envisioned that effective treatment canencompass administering therapeutic agents of the present invention viaoral administration, topical administration, via injection,intranasally, rectally, transdermally, via an impregnated or coateddevice such as an ocular insert or implant, or iontophoretically,amongst other routes of administration.

For administration via injection, the pharmaceutical composition can beinjected intramuscularly, intra-arterially, subcutaneously, orintravenously. A pump mechanism may be employed to administer thepharmaceutical composition over a preselected period. For someembodiments of the invention it is desirable to deliver drug locally,thus injections may be made periocularly, intraocularly,subconjunctively, retrobulbarly, or intercamerally. For some embodimentsof the invention, systemic delivery is preferred.

For systemic administration, the compounds of the invention can beformulated for and administered orally. For administration that mayresult in either regional or systemic distribution of the therapeuticagents, the composition of the invention may be administeredintranasally, transdermally, or via some forms of oral administration,e. g. with use of a mouthwash or lozenge incorporating a compound of theinvention that is poorly absorbed from the G.I. For administration thatmay result in regional or local delivery of the composition of theinvention, iontophoretic or topical administration may be used.

Additionally, the pharmaceutical compositions of the present inventionmay be administered to the ocular surface via a pump-catheter system, orreleased from within a continuous or selective release device such as,e.g., membranes such as, but not limited to, those employed in theOcusert™ System (Alza Corp, Palo Alto, Calif.). The pharmaceuticalcompositions can be incorporated within, carried by or attached tocontact lenses which are then worn by the subject. The pharmaceuticalcompositions can be sprayed onto ocular surface.

The pharmaceutical compositions of the invention may be administered incombination with other therapies for the treatment of the disorder orunderlying disease. For example, the LFA-1 antagonist of the inventionis administered at the same time, or separately during the treatmentperiod for which a subject receives immunosuppressive therapies, such asazathioprine, cyclophosphoramide, methotrextate, antimalarial drugs,mycophenolan mofetile, daclizumab, intravenous immunoglobin therapy, andthe like. In another example, the LFA-1 antagonist of the invention isadministered at the same time or separately during the treatment periodfor which a subject receives other anti-inflammatory treatments, such ascyclosporin A, corticosteroids, NSAIDS, asprin, doxycycline, and thelike. In a further example, the LFA-1 antagonist of the invention isadministered at the same time or separately during the treatment periodfor which a subject receives hormone therapy, and the like. In yet afurther example, the LFA-1 antagonist of the invention is administeredat the same time or separately during the treatment period for which asubject receives anti-allergy therapy, palliative care for dry eyeincluding artificial tears or artificial saliva, muscarinic M3 receptoragonists to increase aqueous secretions, autologous serum, sodiumhyaluronate drops, and the like. These examples are illustrative onlyand are not meant to limit the invention. In some embodiments, the LFA-1antagonist is administered in a single dose. A single dose of a LFA-1antagonist may also be used when it is co-administered with anothersubstance (e.g., an analgesic) for treatment of an acute condition.

In some embodiments, the LFA-1 antagonist (by itself or in combinationwith other drugs) is administered in multiple doses. Dosing may be aboutonce, twice, three times, four times, five times, six times, seventimes, eight times, nine times, ten times or more than ten times perday. Dosing may be about once a month, once every two weeks, once aweek, or once every other day. In one embodiment the drug is ananalgesic. In another embodiment the LFA-1 antagonist and anothertherapeutic substance are administered together about once per day toabout 10 times per day. In another embodiment the administration of theLFA-1 antagonist and another therapeutic substance continues for lessthan about 7 days. In yet another embodiment the co-administrationcontinues for more than about 6, 10, 14, 28 days, two months, sixmonths, or one year. In some cases, co-administered dosing is maintainedas long as necessary, e.g., dosing for chronic inflammationAdministration of the compositions of the invention may continue as longas necessary. In some embodiments, a composition of the invention isadministered for more than 1, 2, 3, 4, 5, 6, 7, 14, or 28 days. In someembodiments, a composition of the invention is administered for lessthan 28, 14, 7, 6, 5, 4, 3, 2, or 1 day. In some embodiments, acomposition of the invention is administered chronically on an ongoingbasis, e.g., for the treatment of chronic pain.

Dosing for the LFA-1 antagonist in the method of the invention may befound by routine experimentation. The daily dose can range from about1×10⁻⁷ g to 5000 mg. Daily dose range may depend on the form of LFA-1antagonist e.g., the esters or salts used, and/or route ofadministration, as described herein. For example, for systemicadministration, typical daily dose ranges are, e.g. about 1-5000 mg, orabout 1-3000 mg, or about 1-2000 mg, or about 1-1000 mg, or about 1-500mg, or about 1-100 mg, or about 10-5000 mg, or about 10-3000 mg, orabout 10-2000 mg, or about 10-1000 mg, or about 10-500 mg, or about10-200 mg, or about 10-100 mg, or about 20-2000 mg or about 20-1500 mgor about 20-1000 mg or about 20-500 mg, or about 20-100 mg, or about50-5000 mg, or about 50-4000 mg, or about 50-3000 mg, or about 50-2000mg, or about 50-1000 mg, or about 50-500 mg, or about 50-100 mg, about100-5000 mg, or about 100-4000 mg, or about 100-3000 mg, or about100-2000 mg, or about 100-1000 mg, or about 100-500 mg. In someembodiments, the daily dose of LFA-1 antagonist is about 100, 200, 300,400, 500, 600, 700, 800, 900, or 1000 mg. In some embodiments, the dailydose of the LFA-1 antagonist is 10 mg. In some embodiments, the dailydose of the LFA-1 antagonist is 100 mg. In some embodiments, the dailydose of LFA-1 antagonist is 500 mg. In some embodiments, the daily doseof LFA-1 antagonist is 1000 mg.

For topical delivery to the ocular surface, the typical daily doseranges are, e.g. about 1×10⁻⁷ g to 5.0 g, or about 1×10⁻⁷ g to 2.5 g, orabout 1×10⁻⁷ g to 1.00 g, or about 1×10⁻⁷ g to 0.5 g, or about 1×10⁻⁷ gto 0.25 g, or about 1×10⁻⁷ g to 0.1 g, or about 1×10⁻⁷ g to 0.05 g, orabout 1×10⁻⁷ g to 0.025 g, or about 1×10⁻⁷ g to 1×10⁻² g, or about1×10⁻⁷ g to 5×10⁻³ g, or about 1×10⁻⁷ g to 2.5×10⁻³ g, or about 1×10⁻⁷ gto 1×10⁻³ g, or about 1×10⁻⁷ g to 5×10⁻⁴ g, or about 1×10⁻⁶ g to 5.0 g,or about 1×10⁻⁶ g to 2.5 g, or about 1×10⁻⁶ g to 1 g, or about 1×10⁻⁶ gto 0.5 g, or about 1×10⁻⁶ g to 0.25 g, or about 1×10⁻⁶ g to 0.1 g, orabout 1×10⁻⁶ g to 5×10⁻² g, or about 1×10⁻⁶ g to 5×10⁻² g, or about1×10⁻⁶ g to 2.5×10⁻² g, or about 1×10⁻⁶ g to 1×10⁻² g, or about 1×10⁻⁶ gto 5×10⁻³ g, or about 1×10⁻⁶ g to 2.5×10⁻³ g, or about 1×10⁻⁶ g to1×10⁻³ g, or about 1×10⁻⁶ g to 5×10⁻⁴ g, or about 1×10⁻⁵ g to 5 g, orabout 1×10⁻⁵ g to 2.5 g, or about 1×10⁻⁵ g to 1 g, or about 1×10⁻⁵ g to0.5 g, or about 1×10⁻⁵ g to 0.25 g, or about 1×10⁻⁵ g to 0.1 g, or about1×10⁻⁵ g to 0.05 g, or about 1×10⁻⁵ g to 2.5×10⁻² g, or about 1×10⁻⁵ gto 1×10⁻² g, or about 1×10⁻⁵ g to 5×10⁻³ g, or about 1×10⁻⁵ g to2.5×10⁻³ g, or about 1×10⁻⁵ g to 1×10⁻³ g, or about 1×10⁻⁵ g to 5×10⁻⁴g. In some embodiments, the daily dose of LFA-1 antagonist is about1×10⁻⁷, 1×10⁻⁶, 1×10⁻⁵, 1×10⁻⁴, 1×10⁻³ g, 1×10⁻² g, 1×10¹ g, or 1 g. Insome embodiments, the daily dose of the LFA-1 antagonist is 1×10⁻⁷ g. Insome embodiments, the daily dose of the LFA-1 antagonist is 1×10⁻⁵ g. Insome embodiments, the daily dose of LFA-1 antagonist is 1×10⁻³ g. Insome embodiments, the daily dose of LFA-1 antagonist is 1×10⁻² g. Insome embodiments the individual dose ranges from about 1×10⁻⁷ g to 5.0g, or about 1×10⁻⁷ g to 2.5 g, or about 1×10⁻⁷ g to 1.00 g, or about1×10⁻⁷ g to 0.5 g, or about 1×10⁻⁷ g to 0.25 g, or about 1×10⁻⁷ g to 0.1g, or about 1×10⁻⁷ g to 0.05 g, or about 1×10⁻⁷ g to 0.025 g, or about1×10⁻⁷ g to 1×10⁻² g, or about 1×10⁻⁷ g to 5×10⁻³ g, or about 1×10⁻⁷ gto 2.5×10⁻³ g, or about 1×10⁻⁷ g to 1×10⁻³ g, or about 1×10⁻⁷ g to5×10⁻⁴ g, or about 1×10⁻⁶ g to 5.0 g, or about 1×10⁻⁶ g to 2.5 g, orabout 1×10⁻⁶ g to 1 g, or about 1×10⁻⁶ g to 0.5 g, or about 1×10⁻⁶ g to0.25 g, or about 1×10⁻⁶ g to 0.1 g, or about 1×10⁻⁶ g to 5×10⁻² g, orabout 1×10⁻⁶ g to 5×10⁻² g, or about 1×10⁻⁶ g to 2.5×10⁻² g, or about1×10⁻⁶ g to 1×10⁻² g, or about 1×10⁻⁶ g to 5×10⁻³ g, or about 1×10⁻⁶ gto 2.5×10⁻³ g, or about 1×10⁻⁶ g to 1×10⁻³ g, or about 1×10⁻⁶ g to5×10⁻⁴ g, or about 1×10⁻⁵ g to 5 g, or about 1×10⁻⁵ g to 2.5 g, or about1×10⁻⁵ g to 1 g, or about 1×10⁻⁵ g to 0.5 g, or about 1×10⁻⁵ g to 0.25g, or about 1×10⁻⁵ g to 0.1 g, or about 1×10⁻⁵ g to 0.05 g, or about1×10⁻⁵ g to 2.5×10⁻² g, or about 1×10⁻⁵ g to 1×10⁻² g, or about 1×10⁻⁵ gto 5×10⁻³ g, or about 1×10⁻⁵ g to 2.5×10⁻³ g, or about 1×10⁻⁵ g to1×10⁻³ g, or about 1×10⁻⁵ g to 5×10⁻⁴ g. In some embodiments, theindividual doses as described above, is repeated 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 times per day.

For other forms of administration, the daily dosages may range about therange described for systemic administration or may range about the rangedescribed for topical administration.

IV. Formulations

The compounds of the invention may be formulated as a sterile solutionor suspension, in suitable vehicles, well known in the art. Suitableformulations and additional carriers are described in Remington “TheScience and Practice of Pharmacy” (20^(th) Ed., Lippincott Williams &Wilkins, Baltimore Md.), the teachings of which are incorporated byreference in their entirety herein.

For injectable formulations, the vehicle may be chosen from those knownin art to be suitable, including aqueous solutions or oil suspensions,or emulsions, with sesame oil, corn oil, cottonseed oil, or peanut oil,as well as elixirs, mannitol, dextrose, or a sterile aqueous solution,and similar pharmaceutical vehicles.

The concentration of drug may be adjusted, the pH of the solutionbuffered and the isotonicity adjusted to be compatible with intravenousinjection, as is well known in the art.

Oral formulations can be tablets, capsules, troches, pills, wafers,chewing gums, lozenges, aqueous solutions or suspensions, oilysuspensions, syrups, elixirs, or dispersible powders or granules, andthe like and may be made in any way known in the art. Oral formulationsmay also contain sweetening, flavoring, coloring and preservativeagents. Pharmaceutically acceptable excipients for tablet forms maycomprise nontoxic ingredients such as inert diluents, such as calciumcarbonate, sodium carbonate, lactose, calcium phosphate, or sodiumphosphate, and the like.

In the case of tablets for oral use, carriers which are commonly usedinclude lactose and corn starch, and lubricating agents such asmagnesium stearate are commonly added. For oral administration incapsule form, useful carriers include lactose and corn starch. Furthernonlimiting examples of carriers and excipients include milk, sugar,certain types of clay, gelatin, stearic acid or salts thereof, calciumstearate, talc, vegetable fats or oils, gums and glycols.

Surfactant which can be used to form pharmaceutical compositions anddosage forms of the invention include, but are not limited to,hydrophilic surfactants, lipophilic surfactants, and mixtures thereof.That is, a mixture of hydrophilic surfactants may be employed, a mixtureof lipophilic surfactants may be employed, or a mixture of at least onehydrophilic surfactant and at least one lipophilic surfactant may beemployed.

A suitable hydrophilic surfactant may generally have an HLB value of atleast 10, while suitable lipophilic surfactants may generally have anHLB value of or less than about 10. An empirical parameter used tocharacterize the relative hydrophilicity and hydrophobicity of non-ionicamphiphilic compounds is the hydrophilic-lipophilic balance (“HLB”value). Surfactants with lower HLB values are more lipophilic orhydrophobic, and have greater solubility in oils, while surfactants withhigher HLB values are more hydrophilic, and have greater solubility inaqueous solutions. Hydrophilic surfactants are generally considered tobe those compounds having an HLB value greater than about 10, as well asanionic, cationic, or zwitterionic compounds for which the HLB scale isnot generally applicable. Similarly, lipophilic (i.e., hydrophobic)surfactants are compounds having an HLB value equal to or less thanabout 10. However, HLB value of a surfactant is merely a rough guidegenerally used to enable formulation of industrial, pharmaceutical andcosmetic emulsions.

Hydrophilic surfactants may be either ionic or non-ionic. Suitable ionicsurfactants include, but are not limited to, alkylammonium salts;fusidic acid salts; fatty acid derivatives of amino acids,oligopeptides, and polypeptides; glyceride derivatives of amino acids,oligopeptides, and polypeptides; lecithins and hydrogenated lecithins;lysolecithins and hydrogenated lysolecithins; phospholipids andderivatives thereof; lysophospholipids and derivatives thereof;carnitine fatty acid ester salts; salts of alkylsulfates; fatty acidsalts; sodium docusate; acyl lactylates; mono- and di-acetylatedtartaric acid esters of mono- and di-glycerides; succinylated mono- anddi-glycerides; citric acid esters of mono- and di-glycerides; andmixtures thereof.

Within the aforementioned group, preferred ionic surfactants include, byway of example: lecithins, lysolecithin, phospholipids,lysophospholipids and derivatives thereof; carnitine fatty acid estersalts; salts of alkylsulfates; fatty acid salts; sodium docusate; acyllactylates; mono- and di-acetylated tartaric acid esters of mono- anddi-glycerides; succinylated mono- and di-glycerides; citric acid estersof mono- and di-glycerides; and mixtures thereof.

Ionic surfactants may be the ionized forms of lecithin, lysolecithin,phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol,phosphatidic acid, phosphatidylserine, lysophosphatidylcholine,lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidicacid, lysophosphatidylserine, PEG-phosphatidylethanolamine,PVP-phosphatidylethanolamine, lactylic esters of fatty acids,stearoyl-2-lactylate, stearoyl lactylate, succinylated monoglycerides,mono/diacetylated tartaric acid esters of mono/diglycerides, citric acidesters of mono/diglycerides, cholylsarcosine, caproate, caprylate,caprate, laurate, myristate, palmitate, oleate, ricinoleate, linoleate,linolenate, stearate, lauryl sulfate, teracecyl sulfate, docusate,lauroyl carnitines, palmitoyl carnitines, myristoyl carnitines, andsalts and mixtures thereof.

Hydrophilic non-ionic surfactants may include, but not limited to,alkylglucosides; alkylmaltosides; alkylthioglucosides; laurylmacrogolglycerides; polyoxyalkylene alkyl ethers such as polyethyleneglycol alkyl ethers; polyoxyalkylene alkylphenols such as polyethyleneglycol alkyl phenols; polyoxyalkylene alkyl phenol fatty acid esterssuch as polyethylene glycol fatty acids monoesters and polyethyleneglycol fatty acids diesters; polyethylene glycol glycerol fatty acidesters; polyglycerol fatty acid esters; polyoxyalkylene sorbitan fattyacid esters such as polyethylene glycol sorbitan fatty acid esters;hydrophilic transesterification products of a polyol with at least onemember of the group consisting of glycerides, vegetable oils,hydrogenated vegetable oils, fatty acids, and sterols; polyoxyethylenesterols, derivatives, and analogues thereof; polyoxyethylated vitaminsand derivatives thereof; polyoxyethylene-polyoxypropylene blockcopolymers; and mixtures thereof; polyethylene glycol sorbitan fattyacid esters and hydrophilic transesterification products of a polyolwith at least one member of the group consisting of triglycerides,vegetable oils, and hydrogenated vegetable oils. The polyol may beglycerol, ethylene glycol, polyethylene glycol, sorbitol, propyleneglycol, pentaerythritol, or a saccharide.

Other hydrophilic-non-ionic surfactants include, without limitation,PEG-10 laurate, PEG-12 laurate, PEG-20 laurate, PEG-32 laurate, PEG-32dilaurate, PEG-12 oleate, PEG-15 oleate, PEG-20 oleate, PEG-20 dioleate,PEG-32 oleate, PEG-200 oleate, PEG-400 oleate, PEG-15 stearate, PEG-32distearate, PEG-40 stearate, PEG-100 stearate, PEG-20 dilaurate, PEG-25glyceryl trioleate, PEG-32 dioleate, PEG-20 glyceryl laurate, PEG-30glyceryl laurate, PEG-20 glyceryl stearate, PEG-20 glyceryl oleate,PEG-30 glyceryl oleate, PEG-30 glyceryl laurate, PEG-40 glyceryllaurate, PEG-40 palm kernel oil, PEG-50 hydrogenated castor oil, PEG-40castor oil, PEG-35 castor oil, PEG-60 castor oil, PEG-40 hydrogenatedcastor oil, PEG-60 hydrogenated castor oil, PEG-60 corn oil, PEG-6caprate/caprylate glycerides, PEG-8 caprate/caprylate glycerides,polyglyceryl-10 laurate, PEG-30 cholesterol, PEG-25 phyto sterol, PEG-30soya sterol, PEG-20 trioleate, PEG-40 sorbitan oleate, PEG-80 sorbitanlaurate, polysorbate 20, polysorbate 80, POE-9 lauryl ether, POE-23lauryl ether, POE-10 oleyl ether, POE-20 oleyl ether, POE-20 stearylether, tocopheryl PEG-100 succinate, PEG-24 cholesterol,polyglyceryl-10oleate, Tween 40, Tween 60, sucrose monostearate, sucrosemonolaurate, sucrose monopalmitate, PEG 10-100 nonyl phenol series, PEG15-100 octyl phenol series, and poloxamers.

Suitable lipophilic surfactants include, by way of example only: fattyalcohols; glycerol fatty acid esters; acetylated glycerol fatty acidesters; lower alcohol fatty acids esters; propylene glycol fatty acidesters; sorbitan fatty acid esters; polyethylene glycol sorbitan fattyacid esters; sterols and sterol derivatives; polyoxyethylated sterolsand sterol derivatives; polyethylene glycol alkyl ethers; sugar esters;sugar ethers; lactic acid derivatives of mono- and di-glycerides;hydrophobic transesterification products of a polyol with at least onemember of the group consisting of glycerides, vegetable oils,hydrogenated vegetable oils, fatty acids and sterols; oil-solublevitamins/vitamin derivatives; and mixtures thereof. Within this group,preferred lipophilic surfactants include glycerol fatty acid esters,propylene glycol fatty acid esters, and mixtures thereof, or arehydrophobic transesterification products of a polyol with at least onemember of the group consisting of vegetable oils, hydrogenated vegetableoils, and triglycerides.

Surfactants may be used in any formulation of the invention where itsuse is not otherwise contradicted. In some embodiments of the invention,the use of no surfactants or limited classes of surfactants arepreferred.

When formulating compounds of the invention for oral administration, itmay be desirable to utilize gastroretentive formulations to enhanceabsorption from the gastrointestinal (GI) tract. A formulation which isretained in the stomach for several hours may release compounds of theinvention slowly and provide a sustained release that may be preferredin some embodiments of the invention. Disclosure of suchgastro-retentive formulations are found in Klausner, E. A.; Lavy, E.;Barta, M.; Cserepes, E.; Friedman, M.; Hoffman, A. 2003 “Novelgastroretentive dosage forms: evaluation of gastroretentivity and itseffect on levodopa in humans” Pharm. Res. 20, 1466-73, Hoffman, A.;Stepensky, D.; Lavy, E.; Eyal, S. Klausner, E.; Friedman, M. 2004“Pharmacokinetic and pharmacodynamic aspects of gastroretentive dosageforms” Int. J. Pharm. 11, 141-53, Streubel, A.; Siepmann, J; Bodmeier,R_(x); 2006 “Gastroretentive drug delivery systems” Expert Opin. DrugDeliver. 3, 217-3, and Chavanpatil, M. D.; Jain, P.; Chaudhari, S.;Shear, R_(x); Vavia, P. R. “Novel sustained release, swellable andbioadhesive gastroretentive drug delivery system for olfoxacin” Int. J.Pharm. 2006 epub Mar. 24. Expandable, floating and bioadhesivetechniques may be utilized to maximize absorption of the compounds ofthe invention.

Intranasal administration may utilize an aerosol suspension ofrespirable particles comprised of the compounds of the invention, whichthe subject inhales. The compound of the invention are absorbed into thebloodstream via pulmonary absorption or contact the lacrimal tissues vianasolacrimal ducts, and subsequently be delivered to the lacrimaltissues in a pharmaceutically effective amount. The respirable particlesmay be solid or liquid, with suitably sized particles, as is known inthe art to be effective for absorption. Compositions for inhalation orinsufflation include solutions and suspensions in pharmaceuticallyacceptable, aqueous or organic solvents, or mixtures thereof, andpowders. The liquid or solid compositions may contain suitablepharmaceutically acceptable excipients as described supra. Preferablythe compositions are administered by the oral or nasal respiratory routefor local or systemic effect. Compositions in preferablypharmaceutically acceptable solvents may be nebulized by use of inertgases. Nebulized solutions may be inhaled directly from the nebulizingdevice or the nebulizing device may be attached to a face mask tent, orintermittent positive pressure breathing machine. Solution, suspension,or powder compositions may be administered, preferably orally ornasally, from devices that deliver the formulation in an appropriatemanner.

For transdermal administration, any suitable formulation known in theart may be utilized, either as a solution, suspension, gel, powder,cream, oil, solids, dimethylsulfoxide (DMSO)-based solutions orliposomal formulation for use in a patch or other delivery system knownin the art. The pharmaceutical compositions also may comprise suitablesolid or gel phase carriers or excipients, which are compounds thatallow increased penetration of, or assist in the delivery of,therapeutic molecules across the stratum corneum permeability barrier ofthe skin. There are many of these penetration-enhancing molecules knownto those trained in the art of topical formulation. Examples of suchcarriers and excipients include, but are not limited to, humectants(e.g., urea), glycols (e.g., propylene glycol), alcohols (e.g.,ethanol), fatty acids (e.g., oleic acid), surfactants (e.g., isopropylmyristate and sodium lauryl sulfate), pyrrolidones, glycerolmonolaurate, sulfoxides, terpenes (e.g., menthol), amines, amides,alkanes, alkanols, water, calcium carbonate, calcium phosphate, varioussugars, starches, cellulose derivatives, gelatin, and polymers such aspolyethylene glycols. The construction and use of transdermal patchesfor the delivery of pharmaceutical agents is well known in the art. See,e.g., U.S. Pat. Nos. 5,023,252, 4,992,445 and 5,001,139. Such patchesmay be constructed for continuous, pulsatile, or on demand delivery ofpharmaceutical agents.

For topical administration, all the formulations for topical ocularadministration used in the field of ophthalmology (e.g., eye drops,inserts, eye packs, impregnated contact lenses, pump delivery systems,dimethylsulfoxide (DMSO)-based solutions suspensions, liposomes, and eyeointment) and all the formulations for external use in the fields ofdermatology and otolaryngology (e.g., ointment, cream, gel, powder,salve, lotion, crystalline forms, foam, and spray) may be utilized as isknown in the art. Additionally all suitable formulations for topicaladministration to skin and mucus membranes of the nasal passages may beutilized to deliver the compounds of the invention. The pharmaceuticalcompositions of the present invention may be a liposomal formulation fortopical or oral administration, any of which are known in the art to besuitable for the purpose of this invention.

Lubricants which can be used to form pharmaceutical compositions anddosage forms of the invention include, but are not limited to, calciumstearate, magnesium stearate, mineral oil, light mineral oil, glycerin,sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid,sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanutoil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, andsoybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, ormixtures thereof. Additional lubricants include, for example, a syloidsilica gel, a coagulated aerosol of synthetic silica, or mixturesthereof. A lubricant can optionally be added, in an amount of less thanabout 1 weight percent of the pharmaceutical composition.

It is envisioned additionally, that the compounds of the invention maybe attached releasably to biocompatible polymers for use in sustainedrelease formulations on, in or attached to inserts for topical orsystemic administration. The controlled release from a biocompatiblepolymer may be utilized with a water soluble polymer to form ainstillable formulation, as well.

Eye drops may be prepared by dissolving the active ingredient in asterile aqueous solution such as physiological saline, bufferingsolution, etc., or by combining powder compositions to be dissolvedbefore use. Other vehicles may be chosen, as is known in the art,including but not limited to: balance salt solution, saline solution,water soluble polyethers such as polyethyene glycol, polyvinyls, such aspolyvinyl alcohol and povidone, cellulose derivatives such asmethylcellulose and hydroxypropyl methylcellulose, petroleum derivativessuch as mineral oil and white petrolatum, animal fats such as lanolin,polymers of acrylic acid such as carboxypolymethylene gel, vegetablefats such as peanut oil and polysaccharides such as dextrans, andglycosaminoglycans such as sodium hyaluronate. If desired, additivesordinarily used in the eye drops can be added. Such additives includeisotonizing agents (e.g., sodium chloride, etc.), buffer agent (e.g.,boric acid, sodium monohydrogen phosphate, sodium dihydrogen phosphate,etc.), preservatives (e.g., benzalkonium chloride, benzethoniumchloride, chlorobutanol, etc.), thickeners (e.g., saccharide such aslactose, mannitol, maltose, etc.; e.g., hyaluronic acid or its salt suchas sodium hyaluronate, potassium hyaluronate, etc.; e.g.,mucopolysaccharide such as chondroitin sulfate, etc.; e.g., sodiumpolyacrylate, carboxyvinyl polymer, crosslinked polyacrylate, polyvinylalcohol, polyvinyl pyrrolidone, methyl cellulose, hydroxy propylmethylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose,hydroxy propyl cellulose or other agents known to those skilled in theart).

The solubility of the components of the present compositions may beenhanced by a surfactant or other appropriate co-solvent in thecomposition. Such cosolvents include polysorbate 20, 60, and 80,Pluronic F68, F-84 and P-103, cyclodextrin, or other agents known tothose skilled in the art. Such co-solvents may be employed at a level offrom about 0.01% to 2% by weight.

The composition of the invention can be formulated as a sterile unitdose type containing no preservatives. The compositions of the inventionmay be packaged in multidose form. Preservatives may be preferred toprevent microbial contamination during use. Suitable preservativesinclude: benzalkonium chloride, thimerosal, chlorobutanol, methylparaben, propyl paraben, phenylethyl alcohol, edetate disodium, sorbicacid, Onamer M, or other agents known to those skilled in the art. Inthe prior art ophthalmic products, such preservatives may be employed ata level of from 0.004% to 0.02%. In the compositions of the presentapplication the preservative, preferably benzalkonium chloride, may beemployed at a level of from 0.001% to less than 0.01%, e.g. from 0.001%to 0.008%, preferably about 0.005% by weight. It has been found that aconcentration of benzalkonium chloride of 0.005% may be sufficient topreserve the compositions of the present invention from microbialattack.

The amount of administration and the number of administrations of theactive ingredient used in the present invention vary according to sex,age and body weight of patient, symptoms to be treated, desirabletherapeutic effects, administration routes and period of treatment. Foreye drops for an adult, the formulations containing the compounds of theinvention may range in concentration from about 0.0001 to 10.0 W/V %,about 0.005 to 10.0 W/V %, about 0.01 to 10.0 W/V %, about 0.05 to 10.0W/V %, about 0.1 to 10.0 W/V %, about 0.5 to 10.0 W/V %, about 1.0 to10.0 W/V %, about 20 to 10.0 W/V %, about 3.0 to 10.0 W/V %, about 4.0to 10.0 W/V %, or about 5.0 to 10.0 W/V %. One embodiment of theinvention has a formulation of about 1.0 to 10.0 W/V % of the compoundsof the invention. One embodiment of the invention has a formulation ofabout 0.01 to 10.0 W/V % of the compounds of the invention. Oneembodiment of the invention has a formulation of about 5.0 to 10.0 W/V %of the compounds of the invention. The administration may beadministered several times a day per eye, preferably one to ten times,more preferably one to four times, most preferably once a day. The sizeof the drop administered may be in the range of about 10-100 μl, about10-90 μl, about 10-80 μl, about 10-70 μl, about 10-60 μl, about 10-50μl, about 10-40 μl, about 10-30 μl, about 20-100 μl, about 20-90 μl,about 20-80 μl, about 20-70 μl, about 20-60 μl, about 20-50 μl, about20-40 μl, or about 20-30 μl. One embodiment of the invention administersa drop in the range of 10-30 □μl. One embodiment of the inventionadministers a drop in the range of 10-100 □μl. One embodiment of theinvention administers a drop in the range of 20-50 μl. One embodiment ofthe invention administers a drop in the range of 10-60 μl.

The formulations of the invention may be administered several drops pertime, one to four drops, preferably one to three drops, more preferablyone to two drops, and most preferably one drop per day.

In formulations for ointment, cream, lotion or spray, the concentrationof the compounds of the invention in the formulations may range about0.0001 10.0 W/V %, about 0.005 to 10.0 W/V %, about 0.01 to 10.0 W/V %,about 0.05 to 10.0 W/V %, about 0.1 to 10.0 W/V %, about 0.5 to 10.0 W/V%, about 1.0 to 10.0 W/V %, about 20 to 10.0 W/V %, about 3.0 to 10.0W/V %, about 4.0 to 10.0 W/V %, or about 5.0 to 10.0 W/V %. Oneembodiment of the invention has a formulation of about 1.0 to 10.0 W/V %of the compounds of the invention. One embodiment of the invention has aformulation of about 0.01 to 10.0 W/V % of the compounds of theinvention. One embodiment of the invention has a formulation of about5.0 to 10.0 W/V % of the compounds of the invention. These formulationsmay be applied or sprayed several times a day, preferably one to sixtimes, more preferably one to four times, and most preferably once aday. The compounding ratio of each ingredient may be suitably increasedor decreased based on the degree of inflammations or infections.

The formulations of the invention can further include otherpharmacological active ingredients as far as they do not contradict thepurpose of the present invention. In a combination of plural activeingredients, their respective contents may be suitably increased ordecreased in consideration of their effects and safety.

V. Kits

The invention also provides kits. The kits include a compound of theinvention in suitable packaging, and written material that can includeinstructions for use, discussion of clinical studies, listing of sideeffects, and the like. The kit may further contain another therapeuticagent that is co-administered with the LFA-1 antagonist of theinvention. In some embodiments, the therapeutic agent and the LFA-1antagonist of the invention are provided as separate compositions inseparate containers within the kit. In some embodiments, the therapeuticagent and the LFA-1 antagonist of the invention are provided as a singlecomposition within a container in the kit. Suitable packaging andadditional articles for use (e.g., measuring cup for liquidpreparations, foil wrapping to minimize exposure to air, dispensers, andthe like) are known in the art and may be included in the kit.

VI. Method to Identify New Compounds Useful in the Method of TreatmentA. Background of the Assay Method

1. Dependence of Ligand Affinities on Divalent Cations

Divalent cations play a critical role in integrin/ligand binding andtheir presence is essential in experimental investigations of theseinteractions. See Hynes, R. O. 1992. Integrins: Versatility, modulation,and signaling in cell adhesion, Cell, 69: 11-25; Humphries, M. J. 1996.Integrin activation: the link between ligand binding and signaltransduction, Curr. Op. Cell Biol., 8: 632-640. The affinities ofICAM-1-Ig and compounds 1, 3, and 4 (structures shown in FIG. 4) forLFA-1 under two sets of commonly used divalent cation conditions weremeasured using fluorescence polarization. The affinity of compound 1 forLFA-1 was first measured in a direct binding assay, and then theaffinities of ICAM-1-Ig and compounds 3 and 4 for LFA-1 were measured incompetition with compound 1 for LFA-1 (FIG. 4, and Table 1 in FIG. 5).The affinity of the A-286982, which binds to the IDAS, was not measuredas it does not compete with compound 1 for binding to LFA-1 (see below)Similar changes in the affinities of compounds 1, 3 and 4 for LFA-1 weremeasured under the different cation conditions as for ICAM-1-Ig. Thesmall molecule affinities increase at least ten-fold in the presence ofMnCl₂ over those measured in CaCl₂ and MgCl₂. These small molecules donot bind to LFA-1 in the absence of divalent cations (data not shown).Similarly, the binding affinities of the soluble protein, ICAM-1-Ig, forLFA-1 in solution, as measured by the same method, in the presence ofMnCl₂, is at least four-fold better than the affinity in the presence ofCaCl₂ and MgCl₂. Thus, unlike the classes of LFA-1 antagonists includingA-286982 that are known to bind to the IDAS region of the I domain (Liu,G., Huth, J. R., Olejniczak, E. T., Mendoza, R., DeVries, P., Leitza,S., Reilly, E. B., Okasinski, G. F., Fesik, S. W., and von Geldern, T.W. 2001. Novel p-arylthio cinnamides as antagonists of leukocytefunction-associated antigen-1/intracellular adhesion molecule-1interaction. 2. Mechanism of inhibition and structure-based improvementof pharmaceutical properties, J. Med. Chem., 44: 1202-1210., Huth, J.R., Olejniczak, E T, Mendoza, R., Liang, H., Harris, E. A. S., Lupher,M. L. Jr., Wilson, A. E., Fesik, S. W., and Staunton, D. E. 2000. NMRand mutagenesis evidence for an I domain allosteric site that regulateslymphocyte function-associated antigen 1 ligand binding, Proc. Natl.Acad. Sci. U.S.A., 97: 5231-5236.) and are reported to bind to LFA-1 ina cation-independent manner (Welzenbach, K., Hommel, U., andWeitz-Schmidt, G. 2002. Small molecule inhibitors induce conformationalchanges in the I domain and the I-like domain of LymphocyteFunction-Associated Antigen-1, J. Biol. Chem., 277: 10590-10598), bothICAM-1-Ig and the class of LFA-1 antagonists represented by compounds1-4 share a divalent cation sensitivity for LFA-1 binding (Table 1).Consequently, in order to identify antagonists of LFA-1/ICAM-1 whichbind in a similar manner to that of ICAM-1-Ig and compounds 1-4, allbinding assays reported herein were performed under similar conditions,in the presence of MnCl₂, which is known to maximize the binding of bothICAM-1 and these cation-sensitive antagonists.

2. Crosslinking of Compound 5 to the αL Subunit of LFA-1

To identify the binding site of small molecule antagonists, compound 5,a tritium-labeled, photoactivatable analogue of compound 3 was bound toLFA-1 and then photocrosslinked. To maximize specific, high affinitycrosslinking, it was necessary to gel filter the samples to removeunbound or weakly bound compound 5 prior to irradiation (FIG. 6, lanes evs. f and g vs. h). In the absence of gel filtration, there wassignificant crosslinking of compound 5 to LFA-1 α subunit, β subunit,and heterodimer (the band at approximately 200,000), whereas nonspecificcrosslinking was not observed in the gel filtered samples (data notshown). Under gel filtration conditions, compound 5 specificallycrosslinked only to the αL subunit (FIG. 6, lanes c and g). Moreover,the presence of compound 3 during the incubation substantially reducedthe incorporation of tritium into the αL subunit (FIG. 6, lane e vs. g).Similarly, in the presence of compound 3, there was a slight reductionof tritium incorporation into the αL subunit, β2 subunit and heterodimerin the absence of gel filtration (FIG. 6, lane f vs. h). No crosslinkingof compound 5 occurred when gel filtered samples of the isolated,structurally intact αL or β2 subunits were used (data not shown). Thus,the high affinity binding site necessary to crosslink after gelfiltration is provided by the intact LFA-1 heterodimer. The absence of ahigh affinity site in the isolated αL subunit is consistent with aprevious study demonstrating lack of interaction of XVA143 with theisolated I domain (Welzenbach et al. 2002).

The site of crosslinking was further defined by fragmenting theaffinity-labeled αL subunit with hydroxylamine, electrophoreticallyseparating the fragments, and then performing N-terminal sequencing onthe radiolabeled fragments to determine their locations within theprotein sequence. Two sequences were identified, the first starting withresidue 1 (sequence found: YNLDVRGARSFS) and the second with residue 30(sequence found: GVIVGAPGEGNST) (Larson, R. S., Corbi, A. L., Berman, L,and Springer, T. 1989. Primary structure of the leukocytefunction-associated molecule-1 alpha subunit: an integrin with anembedded domain defining a protein superfamily, J. Cell Biol., 108:703-712). Both peptides were approximately 500 amino acids long asjudged by their sizes on SDS-PAGE (50-60 kDa); this fragment size isconsistent with the next two predicted cleavage sites (N-G) forhydroxylamine, N507 and N530 (Larson et al. 1989, Bornstein, P. 1969.The nature of a hydroxylamine-sensitive bond in collagen, Biochem.Biophys. Res. Comm., 36: 957-964). No label was incorporated into theC-terminal half of the subunit. Attempts to refine the crosslinking sitefurther were not successful. No definable labeled peptides wererecoverable after limited digestion of the labeled αL subunit witheither cyanogen bromide or Lys-C.

3. Lack of Binding of Compound 2B to LFA-1 Lacking the I Domain

The role of the I domain in the binding of compound 2B and relatedanalogs to LFA-1 was demonstrated by preparing a construct of the αLsubunit lacking the I domain. The 132 construct alone (mock) or togetherwith the construct lacking the I domain or wild type αL was transfectedinto 293 cells, and the binding of compound 2B to the transfected cellswas examined (FIG. 7). Compound 2B showed substantial binding to thewild type αL transfected cells but demonstrated no significant bindingto the cells transfected with αL lacking the I domain relative tobinding to mock (132) transfected cells. Transfectants were also testedfor their ability to adhere to ICAM-1-Ig, and as expected, the LFA-1transfected cells lacking the I domain and mock transfectants showedindistinguishable background levels of binding, while the wild type αLtransfected cells showed robust adhesion (FIG. 7B) (Yalamanchili, P.,Lu, C., Oxvig, C., and Springer T. A. 2000. Folding and function of Idomain-deleted Mac-1 and lymphocyte function-associated antigen-1, J.Biol. Chem., 275: 21877-21882). Evaluation of the binding of a panel ofLFA-1 antibodies to the transfected cells indicated that, apart fromloss of binding by antibodies that mapped to the I domain, the LFA-1heterodimer appeared to be intact in the transfected cells lacking theαL I domain (data not shown).

The data support the conclusion that compound 3 and related moleculesbind to a high affinity site on LFA-1 that overlaps with the ICAM-1binding site which has previously been shown to include the MIDAS motifof the I domain in the αL subunit of LFA-1 (Shimaoka, M., Xiao, T., Liu,J.-H., Yang, Y., Dong, Y., Jun, C-D., McCormack, A. Zhang, R.,Joachimiak, A., Takagi, J., Wang, J.-H., and Springer, T. A. 2003a.Structures of the alpha L I domain and its complex with ICAM-1 reveal ashape-shifting pathway for integrin regulation, Cell, 112: 99-111.).

Corroborating evidence for the close proximity of the ICAM-1 and smallmolecule antagonist binding sites on LFA-1 can be seen in the commoneffect of the deletion of the I domain on the binding of both ICAM-1-Igand compound 2B. Both compound 2B and ICAM-1 were unable to bind toLFA-1 lacking the I domain, the domain in which the ICAM-1 binding siteis located. Moreover, the ability of A-286982 to allosterically modifythe binding of both ICAM-1-Ig and compound 2B is consistent with a closeproximity of their binding sites to the A-286982 binding site in theIDAS motif in the I domain of the LFA-1 α subunit (Liu, G. 200 lb. Smallmolecule antagonists of the LFA-1/ICAM-1 interaction as potentialtherapeutic agents, Expert Opin. Ther. Patents, 11: 1383-1393, Liu etal. 2001). The selective photochemical crosslinking of compound 5 to theα chain of LFA-1 localizes its binding site to within residues 30-507 ofthis subunit. All of the findings noted above are consistent with asingle high affinity small molecule binding site located in the I domainof the cc chain of LFA-1.

Close examination of the photochemical crosslinking study performed witha relatively high concentration of compound 5 (4.1 μM, FIG. 6) affordsdirect evidence for an additional low affinity small molecule bindingsite on LFA-1. Dramatically different protein and crosslinking patternsare observed in the presence and absence of gel filtration. When samplesare gel filtered to remove unbound and weakly bound molecules prior toirradiation, only high affinity labeling of the α subunit is observed.However, in the absence of the gel filtration step, irradiation of thecomplex of compound 5 with LFA-1 results in high intensity crosslinkingto the α subunit and lower intensity crosslinking to a low affinitybinding site in the β subunit whose complex with compound 5 is too weakto survive gel filtration. Under both conditions, the observedcrosslinking is partially inhibited by a large excess (290 μN) ofcompound 3 (FIG. 6, lanes e and g, f and h), demonstrating the specificnature of the binding to both sites. Attempts to crosslink compound 5 toeither of the isolated α orb subunits failed to afford high affinitycomplexes capable of surviving the gel filtration process. Consequently,it appears that the high affinity competitive binding of the class ofcompounds represented by compound 3 requires the presence of an intactfull length LFA-1 heterodimer. Attempts to capture this binding site inconstructs of either of the LFA-1 subunits or the isolated I domainresults in diminished affinity of LFA-1 for ICAM-1 and small moleculeanalogs of compound 3 (e.g. XVA143) (Shimaoka, M., Lu, C., Palframan, R.T., von Andrian, U. H., McCormack, A., Takagi, J., and Springer, T. A.2001. Reversibly locking a protein fold in an active conformation with adisulfide bond: integrin alphaL I domains with high affinity andantagonist activity in vivo, Proc. Natl. Acad. Sci, U.S.A., 98:6009-6014., Welzenbach et al. 2002). It is particularly interesting tonote the presence of a minor LFA-1 heterodimer band that appears in theabsence of gel filtration (FIG. 6, band at >200,000 daltons.) Theintensity of the LFA-1 band as judged by both Coomassie blue stainingand autoradiography is consistent with low affinity binding to a secondsite on the β chain that stabilizes the heterodimer.

It appears, from published gel stabilization studies (Shimaoka, M.,Salas, A., Yang, W., Weitz-Schmidt, G. and Springer, T. 2003b. Smallmolecule integrin antagonists that bind to the β₂ subunit I-like domainand activate signals in one direction and block them in another,Immunity, 19: 391-402, Salas, A., Shimaoka, M., Kogan, A. N., Harwood,C., von Andrian, U. H., and Springer, T. A., 2004. Rolling adhesionthrough an extended conformation of integrin α₁β₂ and relation to α Iand β I-like domain interaction, Immunity, 20: 393-406, Yang, W.,Shimaoka, M., Salas, A., Takagi, J., and Springer, T. A. 2004.Intersubunit signal transmission in integrins by a receptor-likeinteraction with a pull spring. PNAS, 101: 2906-2911), that the bindingsite responsible for the stabilization of LFA-1 to SDS-PAGE resides inthe I-like domain of the β subunit. The data presented herein shows thatthis β subunit binding site is not related to the high affinity bindingsite in the α subunit which is responsible for the direct competitiveinhibition of ICAM-1 binding. However, the β subunit binding siteresponsible for LFA-1 stabilization by compound 3 may be the same as thelow affinity β subunit crosslinking site we have observed.

Overall, the crosslinking and binding experiment results presentedherein indicate that there are two distinct binding sites for the classof LFA-1 small molecule antagonist probes used herein. The first is ahigh affinity binding site in the αL subunit of LFA-1 through which thesmall molecule and LFA-1 form a complex which is stable enough (e.g.K_(d)<25 nM) to survive the gel filtration process. It is this smallmolecule binding site that has been characterized in the bindingexperiments reported here as overlapping the ICAM-1 binding site andthat correlates with: the potent inhibition of LFA-1/ICAM-1 binding bycompounds 3 and 4 (compound 4 IC₅₀=1.4 nM); their potent inhibition ofLFA-1 induced lymphocyte proliferation (compound 4 IC₅₀=3 nM) in vitro;and their inhibition of the immune system's response in vivo (Gadek etal. 2002). The second site is a lower affinity binding site (e.g.K_(d)>1 μM) in the β subunit which is involved with stabilization of theLFA-1 heterodimer under SDS-PAGE. This site is more dynamic by nature(i.e. faster off rate) and does not survive the gelfiltration/photolysis process. The characteristics of this second lowaffinity site are consistent with those of the recently described α/βI-like allosteric antagonist binding site in the I-like domain of the βsubunit (Welzenbach et al. 2002, Shimaoka et al. 2003b, Salas et al.2004, Yang et al. 2004). The low affinity binding of the ICAM-1 mimeticsdescribed herein to the β subunit of LFA-1, presumably to the I-likedomain, is likely due to the sequence homology between the I and I-likedomains, particularly with regard to similarities in MIDAS motifs andtheir affinities for the carboxylic acid moiety common to this class ofantagonists. Given that the β2 family of integrins, including MAC-1,share this subunit, the affinity of compounds for the I-like domain inthe β2 subunit must be attenuated in order to select antagonists whichare specific to LFA-1 (Keating, S., Marsters, J., Beresini, M., Ladner,C., Zioncheck, K., Clark, K., Arellano, F., and Bodary, S. 2000. Puttingthe pieces together: Contribution of fluorescence polarization assays tosmall molecule lead optimization, SPIE Proceedings, 3913: 128-137).

The experiments described above substantiate the high affinity bindingof compounds 3 and 4 to LFA-1 in a manner that is similar to that ofICAM-1, at a site overlapping the ICAM-1 binding site involving theMIDAS motif within the I domain of the LFA-1 α subunit (Shimaoka, M.,Xiao, T., Liu, J.-H., Yang, Y., Dong, Y., Jun, C-D., McCormack, A.Zhang, R., Joachimiak, A., Takagi, J., Wang, J.-H., and Springer, T. A.2003a. Structures of the alpha L I domain and its complex with ICAM-1reveal a shape-shifting pathway for integrin regulation, Cell, 112:99-111.). This is consistent with their proposed mimicry of the ICAM-1epitope (Gadek et al. 2002), and inconsistent with any conclusion thatthey function as α/β I-like allosteric antagonists of LFA-1/ICAM-1(Shimaoka et al. 2003b, Shimaoka, M., and Springer, T. A. 2004.Therapeutic antagonists and the conformational regulation of the β2integrins, Curr. Topics Med. Chem., 4: 1485-1495). The binding of theseICAM-1 mimetics to the β2 integrin subunit, albeit with lower affinity,raises the question of whether ICAM-1 itself binds to a second site inthe I-like domain (Welzenbach et al. 2002, Shimaoka et al. 2003b, Salaset al. 2004, Yang et al. 2004, Shimaoka and Springer 2004) as part of afeedback mechanism. The requirements for divalent cations, to ensure theformation of the active conformation of LFA-1, and physicalcorroboration that probe molecules 1-5, known modulators of LFA-1,compete directly with ICAM-1, are experimental details used in thepresent invention to form a method of identifying new antagonists whichare direct competitive antagonists of LFA-1. The method is useful toidentify new antagonists of LFA-1, to be used in the method of theinvention to treat dry eye disease.

It has been shown, supra, that small molecules can bind with highaffinity to the α-L subunit, which is unique to LFA-1. Consequentlythese compounds can be selective for LFA-1 (αLβ2) over Mac-1(αMβ2). Onepreferred embodiment of the invention is to identify and utilizeselective inhibitors of LFA-1, which may confer advantages intherapeutic safety.

B. Assay Methodology: Competitive Binding Experiments

1. Antagonist Competition in the LFA-1/ICAM-1 and LFA-1/Small MoleculeELISA.

Compounds 2A and 3, A-286982, and sICAM-1 were used to demonstrate themethod. In order to illustrate inhibition of binding of ICAM-1-Ig toLFA-1, these antagonists were titrated into the LFA-1/ICAM-1 ELISA. Theexperiment was performed by the addition of 1/5 serial dilutions ofcompound 3 compound 2A (-σ-), A-286982 (-♦-) and sICAM-1 (-τ-) wereincubated with either ICAM-1-Ig (A) or compound 2B (B) on platescontaining captured LFA-1. The data shown are the average of two platesfrom a single experiment and are representative of several independentmeasurements. The solid lines are the fits of the data. The IC₅₀ values(nM) are provided in the legends.

Typical competition curves for these inhibitors in the ELISA are shownin FIG. 8A. Compound 3 potently inhibited the binding of ICAM-1-Ig toLFA-1 with a 2 nM IC₅₀. Compound 2A, an analogue of compound 3,inhibited binding but with an approximately 10-fold higher IC₅₀ value.A-286982 and sICAM-1 inhibited ICAM-1-Ig binding to LFA-1 but with IC₅₀values that were more than 100-fold that of compound 3.

The ability of these same compounds to inhibit the binding of a FITClabeled small molecule antagonist, compound 2B, to LFA-1 was alsodemonstrated (FIG. 8B). The potencies of compounds 2A and 3 and solubleICAM-1 as inhibitors of compound 2B binding paralleled their potenciesas inhibitors of ICAM-1-Ig binding. Compound 3, compound 2A and sICAM-1inhibited the binding of compound 2B to LFA-1 with IC₅₀ values of 3, 56,and 1200 nM, respectively. A-286982 did not inhibit but rather enhancedthe binding of compound 2B to LFA-1 as indicated by the transientincrease in the absorbance values, reaching a maximal effect atapproximately 4 μM before decreasing.

The evaluation of IC₅₀ values in the LFA-1/small molecule andLFA-1/ICAM-1 ELISAs was extended to a larger set of compounds includinga group of kistrin-derived peptides and small molecules representing theevolution of this class of LFA-1 small molecule antagonists (Gadek etal. 2002). As shown in FIG. 9 (Correlation of IC50 values fromantagonist competition in the LFA-1: ICAM-1 and LFA-1: small moleculesELISAs. The IC₅₀ values of a diverse group of compounds (4 peptides, 5small molecules and sICAM-1) in competition with compound 2B are plottedagainst the IC₅₀ values determined in competition with ICAM-1-Ig forbinding to LFA-1. The slope of the plot is 0.964, y-intercept, 0.237 andR=0.940. Each data point is the average of IC₅₀ values from two plates),there is a good correlation (R=0.94) between the IC₅₀ values forcompetition in each of the two ligand binding assays for this diverseset of compounds, including sICAM-1, compounds 2A and 3, across five logunits of potency. The common trend in potencies between the twoantagonist competition ELISAs with ICAM-1-Ig and compound 2B as ligandsreveals that each compound disrupts the binding of both ICAM-1 and smallmolecule ligands in a mechanistically similar fashion. This parallel inpotency of inhibition demonstrates that ICAM-1-Ig and compound 2B arebinding to the same site on LFA-1 (Wong, A., Hwang, S. M., Johanson, K.,Samanen, J., Bennett, D., Landvatter, S. W., Chen, W., Heys, J. R., Ali,F. E., Ku, T. W., Bondinell, W., Nichols, A. J., Powers, D. A., andStadel, J. M. 1998. Binding of [3H]-SK&F 107260 and [3H]-SB 214857 topurified integrin alphaIIbbeta3: evidence for a common binding site forcyclic arginyl-glycinyl-aspartic acid peptides and nonpeptides, J.Pharmacol. Exp. Therapeutics, 285: 228-235).

2. Antagonist Modulation of Ligand Binding in LFA-1/ICAM-1 andLFA-1/Small Molecule ELISAs.

An antagonist, which inhibits through direct competition with the ligandof interest, exhibits a non-saturable rightward shift of the ligandbinding curves to higher apparent EC₅₀ values with increasing antagonistconcentration and no reduction in the maximal binding of the ligand(Lutz, M., and Kenakin, T 1999. Quantitative Molecular Pharmacology andInformatics in Drug Discovery, John Wiley & Sons, Ltd., New York, Pratt,W. B., and Taylor, P. 1990. Principles of Drug Action: The Basis ofPharmacology, Churchill Livingstone, New York Matthews, J. C. 1993.Fundamentals of Receptor, Enzyme, and Transport Kinetics, CRC Press,Boca Raton, Kenakin, T 1997. Pharmacologic Analysis of Drug-ReceptorInteraction, Lippincott-Raven, Philadelphia) Inhibition will besurmountable but will require increasing amounts of ligand in thepresence of increasing concentrations of a direct competitive inhibitor(Gaddum, J. H., Hameed, K. A., Hathway, D. E., and Stephens, F. F. 1955.Quantitative studies of antagonists for 5-hydroxytryptamine, Q. J. Exp.Physiol., 40: 49-74). The effects of directly competitive compound 3,A-286982 and sICAM-1 on the binding curves of ICAM-1-Ig and compound 2Bto LFA-1 are shown in FIG. 10 as examples of antagonists displayingdirect competition. Titration of ICAM-1-Ig (A, C, E) or compound 2B (B,D, F) in the absence (—⋄—) or presence of antagonist in the LFA-1/ICAM-1and LFA-1/small molecule ELISAs. The antagonists were added in two-folddilutions starting at 2.4 (A) and 2.7 (B) μM sICAM-1, 0.040 (C) and 0.10(D) μM compound 3 and 20 (E) and 50 (F) μM A-286982. The order ofantagonist concentrations was, -□- (lowest added antagonistconcentration), -Δ-, -μ-, -♦-, -▪-, -σ- to -λ- (highest antagonistconcentration). The fits of the data are shown as the solid lines. Thedata shown are from one plate and are representative of a minimum of twoexperiments. (Note that A-286982 (F) resulted in increased binding ofcompound 2B to LFA-1.) In contrast, an allosteric inhibitor may alterthe ligand binding curves by causing a reduction in maximal binding orsaturation in the rightward shifts of the curves (Lutz and Kenakin 1999,Matthews 1993). As shown in FIG. 10A, the presence of increasingconcentrations of sICAM-1 clearly shifted the ICAM-1-Ig binding curvesrightward to higher EC₅₀ values. Additionally, the same maximal extentof binding of ICAM-1-Ig to LFA-1 was observed in the presence andabsence of sICAM-1 as expected when two molecular forms of the samenatural ligand are competing directly for binding to one site on areceptor (Lutz and Kenakin 1999, Pratt, W. B., and Taylor, P. 1990.Principles of Drug Action: The Basis of Pharmacology, ChurchillLivingstone, New York, Matthews 1993, Kenakin, T 1997. PharmacologicAnalysis of Drug-Receptor Interaction, Lippincott-Raven, Philadelphia).Similarly, increasing concentrations of compound 3 also shifted thebinding of ICAM-1-Ig to higher EC₅₀ values with minimal variation inmaximal ICAM-1-Ig binding (FIG. 10C). Although the rightward shifts inthe ligand binding curves in the presence of a competitive antagonistare typically parallel, this is not always the case (Coultrap, S. J.,Sun, H., Tenner, T. E. Jr., and Machu, T. K. 1999. Competitiveantagonism of the mouse 5-hydroxytryptamine3 receptor bybisindolylmaleimide I, a “selective” protein kinase C inhibitor, Journalof Pharmacology and Experimental Therapeutics. 290: 76-82). Thenonparallel slopes for the LFA-1/ICAM-1-Ig binding curves in thepresence and absence of compound 3 may be due to an inability to attaincomplete equilibrium under the heterogeneous ligand binding ELISAconditions with this compound. In the LFA-1/compound 2B format of theligand binding ELISA, increasing concentrations of compound 3 alsoclearly shifted the compound 2B binding curves to higher EC₅₀ valueswith no reduction in maximal binding (FIG. 10D). Increasingconcentrations of sICAM-1 also showed a similar effect (FIG. 10B),although the extent of the shift in the curves was limited by themaximum achievable concentration of sICAM-1 at 2.7 μM. Thus, the effectsof both sICAM-1 and compound 3 on ICAM-1-Ig and compound 2B binding toLFA-1 are characteristic of direct competition as described above.

The effect of A-286982 on ICAM-1-Ig and compound 2B binding to thereceptor was clearly different (FIGS. 10E and 10F). In the LFA-1/ICAM-1ELISA, the ICAM-1-Ig curves were shifted rightward to higher EC₅₀values; however, the maximum binding of ICAM-1-Ig to LFA-1 decreasedconsiderably with increasing concentrations of A-286982. The reductionin maximal binding and rightward shift of the ligand binding curves withincreasing A-286982 concentration are reflective of allostericinhibition as described above. A-286982 causes reductions in both ligandaffinity and binding capacity (Lutz, M., and Kenakin, T 1999.Quantitative Molecular Pharmacology and Informatics in Drug Discovery,John Wiley & Sons, Ltd., New York, Matthews 1993); this demonstratesthat A-286982 is an insurmountable antagonist of ICAM-1-Ig binding. Incontrast, in the LFA/small molecule ELISA, the presence of A-286982 atmicromolar concentrations shifted the compound 2B binding curves tolower EC₅₀ values and appeared to enhance the binding of compound 2B toLFA-1 (FIG. 10F). The contrasting effects of A-286982 on compound 2B andICAM-1-Ig binding may be due to the known allosteric effect of thecompound binding to the IDAS site on LFA-1. The A-286982 binding dataserve as an illustration for allosteric inhibition for small moleculeand protein ligand binding to LFA-1 in the binding experimentsdemonstrated in this method.

Schild analysis can be also used to investigate whether a compoundinhibits ligand binding through direct competition for a single bindingsite (Lutz and Kenakin 1999, Pratt and Taylor 1990, Matthews 1993,Kenakin 1997, Coultrap 1999). This model is based upon the assumptionsthat equiactive responses in an assay are the result of equivalentoccupancy of receptor by ligand and that maximal binding is unchanged bythe presence of antagonist. In a Schild analysis, the dose ratio is theratio of the EC₅₀ values in the presence and absence of antagonist andis a measure of the ligand concentrations leading to equiactiveresponses. This dose ratio was determined for each concentration ofantagonist and the Schild regressions were plotted as shown in FIG. 11.A linear response with a slope of 1 in a Schild regression indicatesthat inhibition by an antagonist is directly competitive and reversible(Lutz and Kenakin 1999, Kenakin 1997). The Schild analysis would yield anonlinear relationship and/or a slope that deviates significantly from 1in the case of an allosteric inhibitor that does not result in areduction of maximal binding (Lutz and Kenakin 1999, Kenakin 1997). TheSchild regressions for both sICAM-1 and compound 3 are shown in FIG. 11with comparable slopes of 1.26 and 1.24, respectively. Schildregressions of s-ICAM-1 (-σ-) and compound 3 (-λ-) antagonism in theLFA-1/ICAM-1 ligand binding ELISA are plotted from the data in FIGS. 5(A) and (C), respectively. The slope of the plot for compound 3 is 1.24with a y-intercept of 10.9 and R=0.99832. The slope of the sICAM-1 plotis 1.26, y-intercept, 8.51 and R=0.99131. Although the Schild analysisrequires a linear regression with a slope close to 1 to demonstratedirect competitive inhibition, there is no guidance in the extensiveliterature as to what range of Schild values are acceptable. Slopes of1.24 and 1.26 fall within the bounds of many published Schild valuesused to support competitive binding conclusions, and therefore, theseslope values are not considered significantly different than 1. Thelinearity of the regression plots and the similarity in slopes of therelationships are consistent with binding of ligand (ICAM-1-Ig) and bothantagonists (sICAM-1 and compound 3) to the same site in a similarmanner.

The binding experiments described above and the analyses discussed areused to form a method for identifying directly competitive inhibitors ofLFA-1. Potentially directly competitive therapeutic agents can beinvestigated using one or more of the experiment types described hereinto ascertain whether the agent of interest does compete with knownnatural and synthetic ligands to compete for binding at the same LFA-1site at which ICAM-1binds. The directly competitive antagonisttherapeutic agents thus identified are used in the method of theinvention to treat a subject in need of treatment for inflammatorydisorders mediated by LFA-1 and its interaction with ICAM-1.

VII. Method of Identifying Compounds Useful in Treating Human Disease

A refined searching method is described herein using the pattern of theinhibition of cell growth by siRNA (small interfering RNA sequences)directed against a cellular target involved in cell growth and humandisease to identify compounds with a similar pattern of cell growthinhibition in a group of cultured cell lines. The use of siRNA data isdesirable because siRNA silences the target's gene and is directlylinked to the inhibition of cell growth by that target. Therefore siRNAdata is useful to correlate the inhibition of a target's function andthe inhibition of cell growth. Compounds identified in this manner areuseful in the treatment of human diseases.

FIG. 12 is a flow chart for the identification of compounds for thetreatment of human diseases using siRNA growth inhibition data.

The method includes choosing a cellular target (for example, a proteinor other biopolymer whose formation is controlled by the transcriptionand/or translation of a gene) involved in the growth of cells containingsaid target whose inhibition would be useful in the control of cellgrowth is selected. This selection can be from lists of such targets inthe public domain including the scientific literature and includesenzymes, receptors and proteins involved in protein-proteininteractions. One such useful target is the association of beta-cateninwith proteins of the TCF family such as TCF-4. These proteins are in theWnt pathway and are involved in the growth and proliferation of a numberof human tumors including common cancer. A compound which binds tobeta-catenin and blocks its association with TCF-4 is useful inpreventing selected gene transcription and the growth of tumors in humancancers, particularly colon cancer. Small interfering RNA (siRNA)sequences unique to the target are purchased from commercial supplierssuch as Dharmacon, Boulder Colo. Cell lines from the National CancerInstitute's panel of 60 cell lines relevant to cancer (for example theColon and Breast derived NCI cell lines) and/or inflammation (forexample the NCI leukemia cell lines) can be grown in the presence ofincreasing amounts of the siRNA directed against the target until thegrowth of the cells is inhibited. Alternatively, a single concentrationof siRNA can be used against all of the cell lines and the relativeinhibition of cell growth can be measured. Cell lines whose growth isdependent on the presence of the target will be inhibited, while othercell lines may be less dependent and consequently their growth will beless inhibited. Thus the inhibition of the panel of the NCI's 60 celllines will produce a pattern of growth inhibition for each siRNA andtarget tested. The use of cell lines not currently in the NCI-60 cellline panel are also envisioned as part of this method. It is envisionedadditionally that reagents, e.g. Lipofectin™ Lipofectamine™, and thelike, can modulate the delivery of siRNA to cells. The NCI's existingdata for dose titration effects of compounds on the growth of the same60 cell lines can be searched using the NCI's COMPARE program or toidentify compounds which have a similar pattern of activity (forexample, the concentration of compound which inhibits the growth ofcells by 50% compared to its uninhibited growth, the GI50 value for acompound). The similarity can be quantified by statistical or othermethods including the Pearson correlation used in the NCI COMPAREprogram. Search algorithms other than NCI COMPARE can be used to definecompound and siRNA similarities. Data from the NCI can be analyzedonline via the world wide web, or it can be downloaded to a computer ornetwork of computers and analyzed offline. Additional databasesincluding public and proprietary databases linking compound structure totheir inhibition of cell growth are also useful for the purposes of thisinvention. For each target, the structures of compounds whose cellgrowth activity pattern is similar to the pattern of growth inhibitionproduced by the siRNA experiment will contain common substructuralfeatures (for example phenyl groups, carboxylic acid groups, hydrogenbond donor group, etc.) which can define a structure activityrelationship (SAR). Such SAR relationships are commonly used bymedicinal chemists skilled in the art of drug discovery to link theactivity of compounds against a target to a common structural motif. Thedevelopment and refinement of an SAR is useful in identifying anddesigning structurally analogous compounds with a probability orlikelihood of showing similar or improved cellular activities. SAR aredeveloped and refined by comparing activities of structurally relatedcompounds. Useful compounds can be synthesized or identified in computersearches of the NCI database or other databases of commerciallyavailable compounds or computer generated libraries of compounds withinteresting and diverse structural features and computed properties(such as ‘druglikeness’). Compounds from commercial or synthetic sourcescan be tested in the cell growth assays for improved potency in theinhibition of cell growth and the data (both for improvements anddeclines in potency) can be used to refine the SAR for inhibition ofcell growth mediated by a target. Iterative cycles of data acquisition,SAR refinement, compound procurement, compound testing/data acquisitioncan identify a compound with a potency below 10 micromolar in theinhibition of cell growth. Such a compound can be useful in drugdiscovery because it is often possible to achieve circulating levels inexcess of 10 micromolar in an animal used as a model of human disease(e.g., a mouse xenograft model of human cancer, as a non-limitingexample). Further testing in animal models relevant to the target forimproved potency, efficacy and duration of action can identify acandidate molecule for the clinical treatment of human diseasesincluding cancer and inflammatory diseases of aberrant cell growth.Alternatively, the identification of compounds which fit an activitypattern opposite of the inhibition of cell growth by siRNA can bestimulants of cell growth useful in diseases and conditions of slow cellgrowth. Enhanced cell growth could be useful in wound healing and otherclinical settings. The method described herein may also utilize thetransfection of a gene for a known protein regulator of the target toaid in identifying a pattern of inhibition sufficiently distinctive tobe able to identify molecules with a similar pattern of activity.

This method is useful in the identification of potent compounds withsignificant potencies below 10 micromolar in the inhibition of cellgrowth. These compounds can be used in animal models of human cancer andinflammation. More preferred are compounds whose inhibition of cellgrowth (GI50) is below 1 micromolar. Even more preferred are compoundswhose growth inhibition (GI50) is less than 100 nM. Most preferred arecompounds with GI50 values below 10 nM. The methods of this inventioncan also be used to identify useful inhibitors of LFA-1, the B-cellreceptor BR3, Grb2 (a protein downstream of growth factor receptors insignaling cascades) and other protein targets inside and outside ofcells. It is particularly useful against targets in the Wnt pathwayincluding beta-catenin for the treatment of human colon cancer. It isalso useful against additional disease related targets in lymphoma,leukemia, colon cancer, melanoma, breast cancer, brain cancer, lungcancer, kidney cancer and other human cancers. The method is useful inidentifying compounds useful in the treatment of human inflammatorydiseases mediated by the growth and proliferation of inflammatory cells.These include but are not limited to Psoriasis, Eczema, Asthma,rheumatoid arthritis and Dry Eye. Compounds which are identified in theabove manner and active in animal models of human disease are useful astreatments of human diseases including cancer and inflammatory diseases.Targets involved in diseases other than cancer and inflammation whichinvolve aberrant cell proliferation can also be used in this method.

Additionally, a method is envisioned to use siRNA cellular activity datafor target or selection of targets by searching public and/orproprietary databases of compound cellular activity for a pattern ofsimilar cellular activity in response to a compound or collection ofcompounds as a method to identify compounds useful in the identificationof a human pharmaceutical.

VIII. Examples

A. Materials

Full length recombinant human membrane-bound LFA-1 and recombinant human5-domain ICAM-1-Ig fusion (ICAM-1-Ig) were produced in human 293 cellsand purified as described (Fisher et al. 1997, Keating et al. 2000).sICAM-1 (a truncated form of native ICAM-1 without the transmembrane andcytoplasmic domains for ease of use in in vitro assays, but with theintact LFA-1 binding epitope) and MEM-48 were from R&D Systems(Minneapolis, Minn.). Mouse monoclonal anti-human β2 integrin (clonePLM2) was generated using standard procedures (Fisher et al. 1997).Small molecules and peptide antagonists were synthesized as described(Gadek et al. 2002, Burdick 1999, Liu et al. 2000). Compounds 1-5 andA-286982 are shown in FIG. 4. Compounds 1, 2A and 2B, are similar tocompound 3 but with the addition of linkers to enable conjugation tofluorescein (compounds 1 and 2B; 2A was not conjugated to fluorescein).Fluorescein conjugates were prepared via coupling of an aminefunctionality with fluorescein-5-isothiocyanate (FITC) (Keating et al.2000). Additional molecules analyzed include compounds 6 and 7 (Gadek etal. 2002), kistrin (Dennis et al. 1990), the non-Kistrin heptapeptides,H₂N—C G F D MP C—CO₂H and H₂N—C G Y^((m))D M P C—CO₂H, cyclic kistrinpeptide C R I P R G D M P D D R C and tetrapeptide, H₂N—C N^((F)) PC—CO₂H, wherein Y^((m)) is meta-tyrosine and N^((F)) isN′-3-phenylpropyl asparagine.

All small molecule antagonists were stored as 10 mM solutions in 50%DMSO at −20° C. Compound 5 was a gift from Hoffman-La Roche Inc.(Nutley, N.J.).

B. Experiments

Example 1: Affinity Measurements

The affinities of the small molecules for LFA-1 were measured usingfluorescence polarization (FP) (Lakowicz 1999, Panvera 1995) in acompetitive format with a small molecule antagonist, compound 1 (FIG.2), as previously described (Keating et al. 2000). All measurements wereperformed in buffer containing 50 mM Hepes, pH 7.2, 150 mM NaCl, 0.05%n-octyglucoside and 0.05% bovine gamma globulins (BGG) and either 1 mMMnCl₂, or 1 mM CaCl₂ and 1 mM MgCl₂. The affinity of compound 1 forLFA-1 was first measured by addition of 2 nM compound 1 to serialdilutions of LFA-1 starting from 1 μM in buffer containing either MnCl₂or CaCl₂ and MgCl₂. Competition experiments were performed by additionof serial dilutions of antagonists to 2 nM compound 1 (using either 3 nMLFA-1 (in MnCl₂) or 40 nM LFA-1 (in CaCl₂ and MgCl₂)). In the ICAM-1-Igcompetition experiments, the LFA-1 concentrations were reduced to 2 and20 nM LFA-1 in the two divalent cation buffer conditions to maximizeinhibition by ICAM-1-Ig. The different LFA-1 concentrations used in theexperiments were taken into account in the affinity calculations (seebelow). The solutions were incubated in 96-well black HE96 plates(Molecular Devices, Sunnyvale, Calif.) for 2 hours at 37° C.Fluorescence Polarization (FP) measurements were performed on an Analystplatereader (Molecular Devices, Sunnyvale, Calif.) using 485 nmexcitation, 530 nm emission and 505 nm dichroic filters. All rawintensity data were corrected for background emissions by subtraction ofthe intensities measured from the appropriate samples withoutcompound 1. The LFA-1 binding and antagonist competition data wereanalyzed using a non linear least squares fit of a four-parameterequation with KaleidaGraph software (Synergy Software, Reading, Pa.) toobtain the EC₅₀ values for the LFA-1 titration and the IC₅₀ values ofthe antagonists. The equation used to fit the data isY=((A−D)/(1+(X/C)̂B))+D, where Y is the assay response, A is Y—value atthe upper asymptote, B is the slope factor, C is the IC₅₀ or EC₅₀ and Dis Y—the value at the lower asymptote. In general, the data measured inboth the homogeneous FP and heterogeneous ELISA formats described below,contain relatively large signal to background ratios and the errorestimates in the fits are typically less than 10% of the final value ofthe fitted parameter. The equilibrium dissociation constants (K_(a)) ofLFA-1 for compound 1 with and without A-286982 were calculated usingKlotz and Hill analyses (Panvera, 1995). The affinities (K_(i)) of theantagonists for LFA-1 were calculated using the IC₅₀ values, the K_(d)of compound 1/LFA-1, and the concentrations of compound 1 and LFA-1 inthe competition experiments (Keating et al. 2000, Jacobs et al. 1975).

Example 2: LFA-1/ICAM-1 and LFA-1/Small Molecule Enzyme-LinkedImmunosorbent Assays (ELISAs)

(A) Antagonist Competition: Small molecules and sICAM-1 were assayed forthe ability to disrupt binding of ICAM-1-Ig or a fluorescein-labeledsmall molecule antagonist, compound 2B, to LFA-1 in a competitive format(Gadek et al. 2002, Burdick 1999, Quan et al. 1998). Compound 2B issimilar to compound 1, but with a longer linker between the smallmolecule and fluorescein to maximize the binding of the anti-fluoresceindetection antibody. 96-well plates were coated with 5 μg/ml (33.3 nM)mouse anti-human β2 integrin (a non-function blocking antibody) inphosphate-buffered saline (PBS) overnight at 4° C. The plates wereblocked with assay buffer (20 mM Hepes, pH 7.2, 140 mM NaCl, 1 mM MnCl₂,0.5% bovine serum albumin (BSA) and 0.05% Tween-20) for 1 hour at roomtemperature. After washing in buffer (50 mM Tris-HCl, pH 7.5, 100 mMNaCl, 1 mM MnCl₂, and 0.05% Tween-20), 8 nM LFA-1 (LFA-1/ICAM-1 ELISA)or 2 nM LFA-1 (LFA-1/small molecule ELISA) were added, followed byincubation for 1 hat 37° C. The plates were washed, and for theLFA-1/ICAM-1 ELISA, serial dilutions of the small molecule antagonistsor sICAM-1 were added to the plates for 30 minutes, followed by additionof 0.89 nM ICAM-1-Ig (final concentration) for 2 hour at 37° C. After anadditional wash, goat anti-hulgG (Fc specific)-HRP was added andincubated for one hour at 37° C. In the LFA-1/small molecule ELISA, thediluted antagonists and 25 nM compound 2B were added concurrently to theplates, followed by a 2-hour incubation at 37° C. Sheepanti-fluorescein-HRP was added after a wash and incubated for one hourat 37° C. For both assays, after washing, the bound HRP-conjugatedantibodies were detected by addition of tetramethylbenzidine (TMB)followed by measurement of the absorbance of the product at 450 nm afterthe addition of 1 M H₃PO₄ to stop the reaction. The IC₅₀ values for eachcurve were determined by fitting to the four-parameter equationdescribed above using KaleidaGraph software. The format and results fromthis form of the LFA-1/ICAM-1 assay are similar to those previouslyreported (Gadek et al. 2002, Burdick 1999); however, this format is morerobust due to antibody capture of the LFA-1 rather than direct coatingonto the ELISA plate.

(B) Ligand Binding: The LFA-1/ICAM-1 and LFA-1/small molecule ELISAswere performed as described above except that serial dilutions of eitherICAM-1-Ig or compound 2B were added to plates either in the presence orabsence of antagonist. In all cases the ligand was added concurrentlywith the antagonist. The plates were incubated for 6 h at 37° C. toapproach equilibrium conditions after antagonist and ligand addition,before wash and addition of the detection antibody. The EC₅₀ values foreach curve were determined by fitting with a four parameter model asdescribed above. The EC₅₀ values generated in the presence and absenceof antagonist were analyzed by Schild regression (Arunlakshana andSchild 1959, Lutz and Kenakin 1999, Pratt and Taylor 1990, Matthews1993, Kenakin, 1997). The Schild plots of Log (Conc. ratio−1) vs.antagonist concentration are calculated from, (Conc. ratio−1)=((ligandEC₅₀ with antagonist)/(ligand EC₅₀ without antagonist))−1. The slopes ofthe plots of the Log (Conc. ratio−1) vs. Antagonist concentration arecalculated by fitting the line to the linear equation, Y=A+BX.

Example 3: Crosslinking of a Radiolabeled, Photoactivatable Analogue ofCompound 3 to LFA-1

Full length human membrane-associated LFA-1 or BSA (0.35 mg/mL [1.4 and5.3 μM, respectively] in 20 mM Hepes, 150 mM NaCl, 5 mM CaCl₂, 5 mMMgCl₂, 1 mM MnCl₂, and 1% n-octylglucoside, pH 7.2) was incubatedovernight at 37° C. with 4.1 μM compound 5, a tritium-labeledphotoactivatable analogue of compound 3 (Kauer et al. 1986), in eitherthe presence or absence of 290 μM compound 3. The molar ratio ofcompound 5 to LFA-1 was 3:1. A 96-well plate precoated with 1% BSA wasused for the incubation. Just prior to crosslinking, excess compound 5was rapidly removed by gel filtration with a G-25 microspin column in a96-well format equilibrated with the same buffer. The LFA-1/compound 5complex was crosslinked by exposure to a high-pressure mercury-vaporlamp (450 watts, Ace glass, Vineland, N.J.). During irradiation, sampleswere cooled on ice and protected by a 5-mm thick plate of borosilicateglass to minimize protein degradation. Residual unlinked compound 5 wasremoved by gel filtration (G-25) as above. The crosslinked complex wasthen denatured in 8 M guanidine hydrochloride (GuHCl) and reduced andalkylated. The treated proteins were subjected to SDS-PAGE followed byCoomassie blue staining. Radiolabeled proteins were visualized byaudioradiography.

To identify compound 5 binding sites, the treated αL and β2 subunitswere separated by size exclusion chromatography in the presence of 6 MGuHCl, 20 mM Hepes, 10 mM EDTA, pH 6.8 and then chemically cleaved with2.6 M hydroxylamine in 10% acetic acid with 7 M GuHCl for 4 h at 75° C.The radiolabeled protein fragments were separated by SDS-PAGE and eithervisualized by autoradiography or transferred onto a polyvinylidenefluoride membrane, stained with Coomassie blue, and then identified byN-terminal protein sequencing.

Example 4: Generation of the αL Construct Lacking the I Domain

The construct used, pLFA.huID.Δp, contains the sequence of the αL genefrom the Nar1 restriction site 5′ of the I domain to the second PflM1restriction site 3′ of the I domain in which the first PflM1 restrictionsite 3′ of the I domain was abolished (Edwards et al. 1995). In order togenerate the mutant lacking the I domain, the following primers weremade: the forward primerCACTGTGGCGCCCTGGTTTTCAGGAAGGTAGTGGATCAGGCACAAGCAAACAGGACCTGACTTC,containing the sequence from the Nar1 site to the start of the I domain,a sequence of DNA encoding GSGSG and the 23 bp of the at sequence afterthe end of the I domain, and the reverse primerTCTGAGCCATGTGCTGGTATCGAGGGGC, which primes at the second PflM1restriction site after the I domain PCR was performed using theseprimers and the pLFA.huID.Δp linearized with Bgl II, which cut at a sitewithin the I domain A DNA fragment was amplified that contained thesequence from the Nar 1 site to the second PflM1 site and in which theentire I domain, from C125 through G311, was replaced with a DNAsequence encoding GSGSG. This piece of DNA was purified, digested withNar1 and PflM1 and inserted into the human αL plasmid (pRKLFAαm) at thecorresponding Nar1 and PflM1 sites. Correct insertion of the DNAsequence encoding GSGSG was confirmed by sequence analysis.

Example 5: Binding of LFA-1 Lacking the I Domain to ICAM-1 or Compound2B

293 cells were transfected with the β2 construct alone (mock) or witheither the wild-type αL construct (wt) or the αL construct lacking the Idomain (I-less) and allowed to recover for 3 days. The cells weredetached and resuspended in adhesion buffer (0.02 M HEPES, pH 7.2, 0.14M NaCl, 0.2% glucose). Binding to plate bound ICAM-1-Ig was performed asdescribed (Edwards et al. 1998). For binding of compound 2B, 2×10⁵ cellswere added per well in a round bottom 96-well plate in adhesion buffercontaining 0.5% BGG, 0.1 mM MnCl₂, 1 μg/ml anti-β2 activating antibodyMEM-48 and 1 μM compound 2B. The cells were incubated for 1 hour at 37°C., washed with cold PBS and fixed with 1% formaldehyde/PBS. The cellswere then incubated with a 1:500 dilution of sheep anti-fluorescein-HRPfor 1 hour at room temperature, washed with PBS and incubated with TMBfor 15 minutes. The reaction was stopped with 1M H₃PO₄ and read at 450nm. In parallel, the transfectants were tested for the structuralintegrity of the surface-expressed αL/β2 complexes and for the presenceor absence of the I domain by FACS analysis using a panel of antibodieswith known binding epitopes (Edwards et al. 1998).

Example 6: Human T-Cell Adhesion Assay (Cell Attachment Assay)

The T-cell adhesion assay is performed using a human T-lymphoid cellline HuT 78. Goat anti-HuIgG (Fc) is diluted to 2 mg/ml in PBS and96-well plates are coated with 50 ml/well at 37° C. for 1 h. Plates arewashed with PBS and blocked for 1 h at room temperature with 1% BSA inPBS. 5 domain ICAM-Ig is diluted to 100 ng/ml in PBS and 50 ml/well wasadded to the plates O/N at 4° C. HuT 78 cells are centrifuged at 100 gand the cell pellet is treated with 5 mM EDTA for about 5 minutes at 37°C. in a 5% CO₂ incubator. Cells are washed in 0.14 M NaCl, 0.02 M Hepes,0.2% glucose and 0.1 mM MnCl₂ (assay buffer) and centrifuged. The cellsare resuspended in assay buffer to 3.0×10⁶ c·ml. Inhibitors are dilutedin assay buffer to a 2× final concentration and pre-incubated with HuT78cells for 30 minutes at room temperature. 100 μl/well of cells andinhibitors are added to the plates and incubated at room temperature for1 h. 100 μl/well of PBS is added and the plates are sealed andcentrifuged inverted at 100 g for 5 minutes. Unattached cells areflicked out of the plate and excess PBS is blotted on a paper towel. 60μl/well p-nitrophenyl n-acetyl-b-D-glucosaminide (0.257 g to 100 mlcitrate buffer) is added to the plate and incubated for 1.5 hat 37° C.The enzyme reaction is stopped with 90 μl/well 50 mM glycione/5 mM EDTAand read on a platereader at 405 nM. HUT 78 cell adhesion to 5dICAM-Igis measured using the p-nitrophenyl method of Langegren, U. (1984). J.Immunol. Methods 57, 379-388.

Example 7: T-Cell Proliferation Assay

This assay is an in vitro model of lymphocyte proliferation resultingfrom activation, induced by engagement of the T-cell receptor and LFA-1,upon interaction with antigen presenting cells. (Springer, et. al. 1990,Nature) Microtiter plates (Nunc 96 well ELISA certified) are pre-coatedovernight at 4° C. with 50 μl of 2 μg/ml of goat anti-human Fc (CaltagH10700) and 50 μl of 0.07 μg/ml monoclonal antibody to CD3 (Immunotech0178) in sterile PBS.

The next day coat solutions are aspirated. Plates are then washed twicewith PBS and 100 μl of 17 ng/ml 5d-ICAM-Ig is added for 4 hours at 37°C. Plates are washed twice with PBS prior to addition of CD4+ T cells.Lymphocytes from peripheral blood are separated from heparinized wholeblood drawn from healthy donors. An alternative method is to obtainwhole blood from healthy donors through leukophoresis. Blood is diluted1:1 with saline, layered, and centrifuged at 2500×g for 30 minutes onLSM (6.2 g Ficoll and 9.4 g sodium diztrizoate per 100 ml) (OrganonTechnica, NJ). Monocytes are depleted using a myeloid cell depletionreagent method (Myeloclear, Labs, Hornby, Ontario, Canada) PBLs areresuspended in 90% heat-inactivated Fetal Bovine serum and 10% DMSO,aliquoted, and stored in liquid nitrogen. After thawing, cells areresuspended in RPMI 1640 medium (Gibco, Grand Island, N.Y.) supplementedwith 10% heat-inactivated Fetal Bovine serum (Intergen, Purchase, N.Y.),1 mM sodium pyruvate, 3 mM L-glutamine, 1 mM nonessential amino acids,500 μg/ml penicillin, 50 μg/ml streptomycin, 50 μg/ml gentamycin(Gibco).

Purification of CD4+ T cells are obtained by negative selection method(Human CD4 Cell Recovery Column Kit # CL110-5 Accurate). 100,000purified CD4+ T cells (90% purity) per microtiter plate well arecultured for 72 hours at 37° C. in 5% CO₂ in 100 ml of culture medium(RPMI 1640 (Gibco) supplemented with 10% heat inactivated FBS(Intergen), 0.1 mM non-essential amino acids, 1 nM Sodium Pyruvate, 100units/ml Penicillin, 100 μg/ml Streptomycin, 50 μg/ml Gentamicin, 10 mMHepes and 2 mM Glutamine) Inhibitors are added to the plate at theinitiation of culture. Proliferative responses in these cultures aremeasured by addition of 1 μCi/well titrated thymidine during the last 6hours before harvesting of cells. Incorporation of radioactive label ismeasured by liquid scintillation counting (Packard 96 well harvester andcounter). Results are expressed in counts per minute (cpm).

Example 8: In-Vitro Mixed Lymphocyte Culture Model

The mixed lymphocyte culture model, which is an in vitro model oftransplantation (A J Cunningham, “Understanding Immunology,Transplantation Immunology” pages 157-159 (1978) examines the effects ofvarious LFA-1 antagonists in both the proliferative and effector arms ofthe human mixed lymphocyte response.

Isolation of Cells: Mononuclear cells from peripheral blood (PBMC) areseparated from heparanized whole blood drawn from healthy donors. Bloodis diluted 1:1 with saline, layered, and centrifuged at ×2500 g for 30minutes on LSM (6.2 g Ficoll and 9.4 g sodium diztrizoate per 100 ml)(Organon Technica, NJ). An alternative method is to obtain whole bloodfrom healthy donors through leukophoresis. PBMCs are separated as above,resuspended in 90% heat inactivated Fetal Bovine serum and 10% DMSO,aliquoted and stored in liquid nitrogen. After thawing, cells areresuspended in RPMI 1640 medium (Gibco, Grand Island, N.Y.) supplementedwith 10% heat-inactivated Fetal Bovine serum (Intergen, Purchase, N.Y.),1 mM sodium pyruvate, 3 mM L-glutamine, 1 mM nonessential amino acids,500 μg/ml penicillin, 50 μg/ml streptomycin, 50 μg/ml gentamycin(Gibco).

Mixed Lymphocyte Response (MLR): One-way human mixed lymphocyte culturesare established in 96-well flat-bottomed microtiter plates. 1.5×10⁵responder PBMCs are co-cultured with an equal number of allogeneicirradiated (3000 rads for 3 minutes, 52 seconds stimulator PBMSc in 200μl of complete medium. LFA-1 antagonists are added at the initiation ofcultures.

Cultures are incubated at 37° C. in 5% CO₂ for 6 days, then pulsed withof ³H-thymidine (6.7 Ci/mmol, NEN, Boston, Mass.) for 6 hours. Culturesare harvested on a Packard cell harvester (Packard, Canberra, Canada)[³H] TdR incorporation is measured by liquid scintillation counting.Results are expressed as counts per minute (cpm).

Example 9: Rabbit Model to Reverse the Onset of Dry Eye

Dry eye is created in rabbits by surgically closing the lacrimal glandexcretory duct, and allowing the rabbits to remain untreated for atleast four weeks. See Gilbard, J. P, 1996 “Dry Eye: pharmacologicalapproaches, effects, and progress” CLAO J. 22, 141-145. After confirmingdry eye by Schirmer test, and ocular surface staining, LFA-1 antagonistsof the invention is instilled as a solution at concentrations of 0.01,0.1, and 1.0% in neutral, isotonic buffered aqueous solution.Administration is one 50 microliter drop to the ocular surface up to 5times a day, every day for 4 weeks. The symptoms of dry eye aremonitored once a week for 4 weeks and an increase in Schirmer scoresand/or a decrease in the amount of ocular surface staining indicates theefficacy of the LFA-1 antagonist in the treatment of dry eye disease.

Example 10: Phase 1 Human Study

Up to 56 healthy individuals are enrolled. A randomized, controlled,dose escalation trial of both single and multiple administrations ofLFA-1 antagonist is conducted. Cohorts of 7 subjects each (5 treatment,2 placebo) are treated at each of 6-8 dose levels of LFA-1 antagonistsformulated as sterile, neutral, isotonic, buffered aqueous solutions.Subjects receive a single intra-ocular administration on Day 1. Samplesare obtained for pharmacokinetic and pharmacodynamic assessments overthe subsequent week. Starting Day 8, subjects receive the same dose ofLFA-1 antagonist daily for a total of 14 days. PK/PD assessments, safetylaboratory studies, Schirmer testing, corneal staining and conjunctivalbiopsies are assessed.

Example 11: Phase II Human Study

150 adult patients with dry eye as defined by key inclusion/exclusioncritieria are enrolled. The patients may or may not have Sjogren'ssyndrome or Sjogren's disease. A randomized, controlled dose findingtrial of LFA-1 antagonists is conducted. Three groups of patientsreceive either Restasis at the labeled dose, or, one of two dose levelsof LFA-1 antagonist, formulated as a neutral, buffered, isotonic aqueoussolution, daily for twelve weeks. Patients are followed for safety andfor evidence of improvement in Schirmer's test, corneal staining andoverall disease severity index for a follow up period of three months.Conjunctival biopsies are obtained in a subset of patients.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1.-97. (canceled)
 98. A method of treating an immune response associatedwith cytokine synthesis in a patient with an LFA-1-mediated disease ordisorder, said method comprising administering to said patient atherapeutically effective amount of the compound

or a pharmaceutically acceptable salt thereof.
 99. A method as definedin claim 98, wherein the patient does not have Sjogren's syndrome. 100.A method as defined in claim 98, wherein the patient has Sjogren'ssyndrome.
 101. A method as defined in claim 98, wherein saidadministration comprises topical administration of said compound via acarrier vehicle selected from a group consisting of liquid drops, liquidwash, gel, ointment, spray, liposome, and any combination of any of theforegoing.
 102. A method as defined in claim 98, wherein saidadministration comprises topical administration of said compound or saltto an eye of said patient via a device selected from the groupconsisting of a pump-catheter system, a continuous or selective releasedevice, a contact lens, or any combination of any of the foregoing. 103.A method as defined in claim 98, wherein said administration comprisessystemically administering a liquid or liquid suspension of saidcompound or salt via nose drops, nasal spray, nebulized liquid, or anycombination of any of the foregoing to oral or nasopharyngeal airways ofsaid patient, such that said inhibition effective amount of saidcompound or salt contacts one or more of the lacrimal gland,conjunctival tissue, tears, or ocular surface of the eye of saidpatient.
 104. A method as defined in claim 98, wherein saidadministration comprises administering an oral form of said compound orsalt, such that said inhibition effective amount of said compound orsalt contacts one or more of the lacrimal gland, conjunctival tissue,tears, or ocular surface of the eye of said patient via systemicabsorption and circulation.
 105. A method as defined in claim 98,wherein said administration comprises administering an injectable formof said compound or salt, such that said inhibition effective amount ofsaid compound or salt contacts one or more of the lacrimal tissues,conjunctival tissue, tears, or ocular surface of the eye of said patientvia systemic absorption and circulation.
 106. A method as defined inclaim 98, wherein said administration comprises administering asuppository dosage form of said compound or salt, such that saidinhibition effective amount of said compound or salt contacts one ormore of the lacrimal tissues, conjunctival tissue, tears, or ocularsurface of the eye of said patient via systemic absorption andcirculation.
 107. A method as defined in claim 98, wherein saidadministration comprises administering by topical instillation to thesurface of the eye a gel, cream, powder, foam, crystal, liposome, spray,liquid, or any combination of any of the foregoing of said compound orsalt.
 108. A method as defined in claim 98, wherein said compound orsalt is administered to the ocular surfaces of said patient in aninhibition effective amount sufficient to achieve concentrations thereofof from about 1×10⁻⁷ to about 1×10⁻¹ moles/liter.
 109. A method oftreating an immune response associated with cytokine synthesis in apatient with an LFA-1-mediated disease or disorder, said methodcomprising administering to the eye of said patient a therapeuticallyeffective amount of the compound

or a pharmaceutically acceptable salt thereof, whereby saidadministration is effective in promoting tear secretion or mucinproduction in said eye in said patient, and wherein said patient doesnot have Sjogren's syndrome.
 110. A method of treating an immuneresponse associated with cytokine synthesis in a patient with anLFA-1-mediated disease or disorder, said method comprising administeringto an eye of said patient a therapeutically effective amount of thecompound

or a pharmaceutically acceptable salt thereof, whereby saidadministration is effective in promoting tear secretion or mucinproduction in said eye in said patient, and wherein said patient hasSjogren's syndrome.
 111. A method of treating an immune responseassociated with cytokine synthesis in a patient with an LFA-1-mediateddisease or disorder, said method comprising performing a dry eyediagnostic test on said patient; determining whether said patientsuffers from a dry eye disease based on the results of said diagnosticstep; and upon diagnosis of said dry eye disease, administering to saidpatient an inhibition effective amount of the compound

or a pharmaceutically acceptable salt thereof, and wherein said patientdoes not have Sjogren's syndrome.
 112. A method of treating an immuneresponse associated with cytokine synthesis in a patient with anLFA-1-mediated disease or disorder, said method comprising performing adry eye diagnostic test on said patient; determining whether saidpatient suffers from a dry eye disease based on the results of saiddiagnostic step; and upon diagnosis of said dry eye disease,administering to said patient an inhibition effective amount of thecompound

or a pharmaceutically acceptable salt thereof, and wherein said patienthas Sjogren's syndrome.
 113. A method as defined in claim 111, whereinsaid diagnostic step comprises imaging an eye of said patient, analysisof a biological sample of an eye of said patient, or a combination ofthe foregoing.
 114. A method as defined in claim 112, wherein saiddiagnostic step comprises imaging an eye of said patient, analysis of abiological sample of an eye of said patient, or a combination of theforegoing.
 115. A method as defined in claim 98, wherein saidadministration is via a sustained release insert or implant,subconjunctival injection, intraocular injection, periocular injection,retrobulbar injection, intracameral injection, or any combination of anyof the foregoing.
 116. A method as defined in claim 98, wherein saidadministration comprises delivery of a liquid or liquid suspension ofsaid compound or salt via nose drops, nasal spray, nebulized liquid, orany combination of any of the foregoing to oral or nasopharyngealairways of said patient, such that said inhibition effective amount ofsaid compound or salt contacts one or more of the lacrimal gland,conjunctival tissue, tears, or ocular surface of the eye of said patientvia nasolacrimal ducts.
 117. A method as defined in claim 98, whereinsaid administration comprises administering an injectable form of saidcompound or salt, such that said inhibition effective amount of saidcompound or salt contacts one or more of the lacrimal tissues,conjunctival tissue, tears, or ocular surface of the eye of said patientvia local delivery.
 118. A method as defined in claim 102, wherein saidcontinuous or selective release device comprises an ocular insert orimplant.
 119. A method as defined in claim 102, wherein said inhibitioneffective amount of said compound or salt is distributed regionally toone or more of the nose, nasal passages, and nasal cavity.
 120. A methodas defined in claim 118, wherein said continuous or selective releasedevice comprises a biocompatible polymer.
 121. A method as defined inclaim 107, wherein said gel, cream, powder, foam, crystals, liposomes,spray, or liquid suspension administers said compound or salt viacontrolled release of said compound or salt from a biocompatiblepolymer.